Therapeutic uses of bactericidal-permeability-increasing protein dimer products

ABSTRACT

Improved therapeutic uses of bactericidal/permeability-increasing protein (BPI) involve use of BPI protein product formulations in the form of physiologically stable dimeric associations of BPI protein product monomers characterized by enhanced in vivo biological activity. Preferred formulations include 50 percent or more by weight dimeric product.

This is a continuation of U.S. application Ser. No. 08/212,132, now U.S.Pat. No. 5,447,913 filed Mar. 11, 1994.

BACKGROUND OF THE INVENTION

The present invention provides bactericidal/permeability-increasingprotein (BPI) products characterized by enhanced in vivo biologicalactivity and stable pharmaceutical compositions containing the same.

Lipopolysacchaxide (LPS) is a major component of the outer membrane ofGram-negative bacteria and consists of serotype-specific O-side-chainpolysaccharides linked to a conserved region of core oligosaccharide andlipid A. Raetz, Ann. Rev. Biochem., 59:129-170 (1990). LPS is animportant mediator in the pathogenesis of Gram-negative septic shock,one of the major causes of death in intensive-care units in the UnitedStates. Morrison, et al., Ann. Rev. Med. 38:417-432 (1987).

LPS-binding proteins have been identified in various mammalian tissues.Morrison, Microb. Pathol., 7:389-398 (1989); Roeder, et al., Infect.,Immun., 57:1054-1058 (1989). Among the most extensively studied of theLPS-binding proteins is bactericidal/permeability-increasing protein(BPI), a basic protein found in the azurophilic granules ofpolymorphonuclear leukocytes. Human BPI protein has been isolated frompolymorphonuclear neutrophils by acid extraction combined with eitherion exchange chromatography Elsbach, J. Biol. Chem., 254:11000 (1979)!or E. coli affinity chromatography Weiss, et al., Blood, 69:652 (1987)!and has potent bactericidal activity against a broad spectrum ofGram-negative bacteria.

The amino acid sequence of the entire human BPI protein, as well as theDNA encoding the protein, have been elucidated in FIG. 1 of Gray, etal., J. Biol. Chem., 264:9505 (1989), incorporated herein by reference(SEQ ID NOs: 1 and 2). The Gray et al. publication discloses theisolation of human BPI-encoding cDNA from a cDNA library derived fromDMSO-induced cells of the human promyelocytic leukemia HL-60 cell line(ATTC CCL 240). Multiple PCR amplifications of DNA from cDNA libraryderived from such DMSO-induced HL-60 cells as well as DNA from normalhuman blood and bone marrow cell have revealed the existence of humanBPI-encoding cDNAs wherein the codon specifying valine at amino acidposition 151 is either GTC (as set out in SEQ ID No: 1) or GTG.Moreover, cDNA species employing GTG to specify valine at position 151have also been found to specify either lysine (AAG) for the position 185amino acid (as in SEQ ID Nos: 1 and 2) or a glutamic acid residue (GAG)at that position.

A proteolytic fragment corresponding to the N-terminal portion of humanBPI holoprotein possesses the antibacterial activity of thenaturally-derived 55 kDa human BPI holoprotein. In contrast to theN-terminal portion, the C-terminal region of the isolated human BPIprotein displays only slightly detectable anti-bacterial activity. Ooi,et al., J. Exp. Med., 174:649 (1991). A BPI N-terminal fragmentdesignated rBPI₂₃, Gazzano-Santoro et al., Infect. Immun. 60:4754-4761(1992), and comprising approximately the first 199 amino acids of thehuman BPI holoprotein, has been produced by recombinant means as a 23 kDprotein.

The bactericidal effect of BPI has been shown to be highly specific forsensitive Gram-negative species. The precise mechanism by which BPIkills bacteria is not yet completely elucidated, but it is known thatBPI must first attach to the surface of susceptible Gram-negativebacteria. This initial binding of BPI to the bacteria involveselectrostatic and hydrophobic interactions between the basic BPI proteinand negatively charged sites on LPS. LPS has been referred to as"endotoxin" because of the potent inflammatory response that itstimulates, i.e., the release of mediators by host inflammatory cellswhich may ultimately result in irreversible endotoxic shock. BPI bindsto lipid A, the most toxic and most biologically active component ofLPS.

In susceptible bacteria, BPI binding is thought to disrupt LPSstructure, leading to activation of bacterial enzymes that degradephospholipids and peptidoglycans, altering the permeability of thecell's outer membrane, and initiating events that ultimately lead tocell death. Elsbach and Weiss, Inflammation: Basic Principles andClinical Correlates, eds. Gallin et al., Chapter 30, Reven Press, Ltd.(1992)!. BPI is thought to act in two stages. The first is a sublethalstage that is characterized by immediate growth arrest, permeabilizationof the outer membrane and selective activation of bacterial enzymes thathydrolyze phospholipids and peptidoglycan. Bacteria at this stage can berescued by growth in serum albumin supplemented media but not by growthin whole blood, plasma or serum. The second stage, defined by growthinhibition that cannot be reversed by serum albumin, occurs afterprolonged exposure of the bacteria to BPI and is characterized byextensive physiologic and structural changes, including penetration ofthe cytoplasmic membrane.

BPI-induced permeabilization of the bacterial cell envelope tohydrophobic probes such as actinomycin D is rapid and depends upon theinitial binding of BPI to LPS, leading to organizational changes whichprobably result from binding to the anionic groups in the KDO region ofLPS, normally responsible for stabilizing the outer membrane throughbinding of Mg⁺⁺ and Ca⁺⁺. Binding of BPI and subsequent bacterialkilling depends, at least in part, upon the LPS polysaccharide chainlength, with long O chain bearing organisms being more resistant to BPIbactericidal effects than short, "rough" organisms (Weiss et al., J.Clin. Invest. 65:619-628 (1980). This first stage of BPI action isreversible upon dissociation of the BPI, a process requiring synthesisof new LPS and the presence of divalent cations (Weiss et al., J.Immunol. 132: 3109-3115 (1984). Loss of bactericidal viability, however,is not reversed by processes which restore the membrane integrity,suggesting that the bactericidal action is mediated by additionallesions induced in the target organism and which may be situated at thecytoplasmic membrane (Mannion et al., J. Clin. Invest. 86: 631-641(1990)). Specific investigation of this possibility has shown that, on amolar basis, BPI is at least as inhibitory of cytoplasmic membranevesicle function as polymyxin B (In't Veld et al., Infection andImmunity 56: 1203-1208 (1988)) but the exact mechanism has not yet beenelucidated.

Heparin is a heterogenous group of straight-chain anionicmucopolysaccharides (glycosaminoglycans) having anticoagulantproperties. Although others may be present, the main sugars occurring inheparin are: (1)α-L-iduronic acid 2-sulfate, (2)2-deoxy-2-sulfamino-α-D-glucose 6-sulfate, (3) β-D-glucuronic acid, (4)2-acetamido-2-deoxy-α-D-glucose, and (5) α-L-iduronic acid. These sugarsare present in decreasing amounts, usually in the order(2)>(1)>(4)>(3)>(5), and are joined by glycosidic linkages, formingpolymers of varying sizes. Heparin is strongly acidic because of itscontent of covalently linked sulfate and carboxylic acid groups. Heparinis found within mast cell granules and is released upon degranulation. Acell associated form of heparin is termed heparan surfate. Heparansulfate is a broad term used to describe a variety of sulfatedproteoglycans (HSPG's) found with a near-ubiquitous distribution onmammalian cell surface membranes and in the extracellular matrix. HSPGcontains a variable percentage of pentamaric heparin-like sequences thatfunction in a similar fashion as soluble heparin. The HSPG's serve as arepository for antithrombin III (ATIII) and for heparin-binding growthfactors such as fibroblast growth factors (FGF) 1-5, IL-8, GM-CSF andIL-3. Folkman et al., Inflammation: Basic Principles and ClinicalCorrelates, 2d Ed. Chapter 40, pp 821-839 (1992). In fact, cells madegenetically deficient in HSPG's require exogenous heparin for growth.

Heparin is commonly administered in doses of up to 400 U/kg duringsurgical procedures such as cardiopulmonary bypass, cardiaccatherization and hemodialysis procedures in order to prevent bloodcoagulation during such procedures. The anticoagulant effect of beparinin blood is a result of the interaction of beparin with ATIII. Theheparin/ATIII complex is a potent inhibition of many of the clottingfactors of the coagulation cascade. Specific inhibition has beendemonstrated for activated Factors IXa, Xa, XIa, XIIIa and thrombin. Theheparin/ATIII complex has the highest affinity for Factor Xa andthrombin which are common to both the intrinsic and extrinsic clottingpathways involved as the last two steps of the clotting cascade thatresults in the conversion of fibrinogen to fibrin. The additionalantibacterial and antiendotoxin effects of BPI would be particularlyadvantageous in post-surgical heparin neutralization.

When heparin is administered for anficoagulant effects during surgery,it is an important aspect of post-surgical therapy that the effects ofheparin are promptly neutralized so that normal coagulation function canbe restored.

Angiogenesis is closely associated with endothelial cell proliferationand constitutes the development of new capillar blood vessels. As such,it is an important process in mammalian development and growth, and inmenstruation processes. The release of angiogenic growth factors, suchas fibroblast growth factors 1-5, induces proliferation of endothelialcells via a heparin-dependent receptor binding mechanism. See Yayon etal., Cell, 64: 841-848 (1991). These heparin-binding growth factors canbe released due to vascular trauma (wound healing), immune stimuli(autoimmune disease), inflammatory mediators (prostaglandins) and fromtumor cells.

Angiogenesis is also associated with a number of pathological conditionsin which it would be desirable to inhibit such new blood vesseldevelopment. As one example, angiogenesis is critical to the growth,proliferation, and metastasis of various tumors. Other pathologicalconditions associated with angiogenesis include diabetic retinopathy,retrolental fibroplasia, neovascular glaucoma, psoriasis, angiofibromas,immune and non-immune inflammation including rheumatoid arthritis,capillary proliferation within atherosclerotic plaques, hemangiomas,endometriosis and Kaposi's Sarcoma.

Chronic inflammation is usually accompanied by angiogenesis. Arthritisis a chronic syndrome characterized by the inflammation of theperipheral joints accompanied by synovial thickeing and the influx ofimmune factors and cells such as polymorphonuclear leukocytes (PMN). Inrheumatoid arthritis, the inflammation is immune driven, while inreactive arthritis, inflammation is associated with infection of thesynovial tissue with pyrogenic bacteria or other infectious agents.Folkman et al., Inflammation: Basic Principles and Clinical Correlates,2d Ed. Chapter 40, pp 821-839 (1992) note that many types of arthritisprogress from a stage dominated by an inflammatory infiltrate in thejoint to a later stage in which a neovascular pannus invades the jointand begins to destroy cartilage. While it is unclear whetherangiogenesis in arthritis is a causative component of the disease, andnot an epiphenomenon, there is evidence that angiogenesis is necessaryfor the maintenance of synovitis in rheumatoid arthritis.

Co-owned, copending U.S. patent application Ser. No. 08/030,644 filedMar. 12, 1993, now U.S. Pat. No. 5,348,942, continuation-in-part U.S.patent application Ser. No. 08/093,202, filed Jul. 15, 1993, nowabandoned, continuation-in-part U.S. patent application Ser. No.08/183,222 filed Jan. 14, 1994, now abandoned, and continuation-in-partU.S. patent application Ser. No. 08/209,762, filed concurrentlyherewith, address use of BPI protein products for treatment ofconditions not directly associated with Gram-negative bacterialinfection, including neutralization of the anti-coagulant properties ofheparin, inhibition of angiogenesis, tumor and endothelial cellproliferation and treatment of chronic inflammatory disease states suchas arthritis.

Various other utilities have been described for therapeuticadministration of BPI protein products. Co-owned, copending U.S. patentapplication Ser. No. 08/031,145, filed Mar. 12, 1993, now abandoned,addresses use of BPI protein products in treatment of mycobacterialdiseases. Co-owned, copending U.S. patent application Ser. No.08/132,510, filed Oct. 5, 1993, now abandoned, addresses use of BPIprotein products in the treatment of conditions involving depressedreticuloendothelial system function. Co-owned, copending U.S. patentapplication Ser. No. 08/125,651, filed Sep. 22, 1993, now abandoned,addresses synergistic combinations of BPI protein products andantibiotics. Co-owned and copending U.S. patent application Ser. No.08/093,201 filed Jul. 14, 1993, now abandoned, addresses methods forpotentiating BPI protein product bactericidal activity by administrationof LBP protein products. Co-owned and copending U.S. patent applicationSer. No. 08/031,144 filed Mar. 12, 1993, now abandoned, andcontinuation-in-part application Ser. No. 08/209,479 filed concurrentlyherewith, addresses administration of BPI protein products for treatmentof Helicobacter infections. Co-owned, copending U.S. patent applicationSer. No. 08/188,221 filed Jan. 24, 1994, now abandoned, incorporated byreference herein, addresses use of BPI protein products in the treatmentof humans exposed to Gram-negative bacterial endotoxin in circulation.The disclosures of the above applications are specifically incorporatedby reference herein.

Efforts to produce pharmaceutical grade BPI products for human treatmenthave not yielded uniformly satisfactory results. A principal reason forthis is the nature of the amino acid sequence of human BPI and thenature of the recombinant host cell environment in which the productsare produced. As one example, biologically-active rBPI products producedas secretory products of CHO host cells transfected with a constructencoding the initial 199 residues of BPI rBPI (1-199)! may be purifiedin good yields. As noted in co-owned, copending U.S. Pat. No. 5,439,807elution of BPI products from S-Sepharose beads incorporated into rollerbottles containing transformed CHO cells yielded substantially monomericBPI products when a 1.0M NaCl-Acetate buffer was employed, but yieldedmultimeric protein forms when a 1.5M NaCl-Acetate buffer was thenemployed. Moreover, secreted expression products resulting from CHOcells transfected with DNA encoding a secretory leader sequence and BPIamino acid residues 1-199 actually yielded mixtures of carboxyterminal-shortened BPI protein products terminating at residue 193 or atother residues intermediate residue 193 and 199. Co-owned, copendingU.S. Pat. No. 5,420,019, addresses analog BPI protein products and DNAsequences encoding the same wherein cysteines at positions 132 or 135are replaced by different amino acids for the purpose of reducingmultimer and cysteine adduct formation in recombinant products and alsoaddresses development of recombinant expression products using DNAsencoding the initial amino terminal residue (1) to from about 175 to 193of BPI, which products display reduced carboxy terminal heterogeneity.

There continues to be a need in the art for improved BPI protein productpreparations. Such products would be obtainable in large yield asrecombinant products from transformed host cells, would retain thebactericidal, LPS-binding, LPS-neutralizing, heparin binding, heparinneutralizing and other biological activities of BPI rendering themsuitable for therapeutic use, and would ideally display enhanced in vivobiological activity, thus providing for improved therapeutic methodsinvolving administration of BPI protein products.

SUMMARY OF THE INVENTION

The present invention provides significant improvements in therapeuticuses of BPI protein products through the provision of productformulations which include BPI protein products in the form ofphysiologically stable covalently or non-covalently linked dimericassociations of BPI protein product monomers ("BPI dimers") which havebeen discovered to possess enhanced in vivo biological activity incomparison to monomeric product forms. In preferred therapeuticformulations of the invention, dimeric forms of BPI protein productsconstitute greater than 50 percent by weight of such products. Becauseof the enhanced in vivo activity of dimeric associations of BPI proteinproduct monomers, product formulations including greater than 75 percentby weight, greater than 90 percent by weight and 95 percent or more byweight. dimeric product are successively more preferred.

Therapeutic BPI protein product formulations of the present inventionpreferably include homodimeric products, but heterodimeric products arealso within the contemplation of the invention. Formulations alsopreferably include dimeric and, if any, monomeric forms of BPI proteinproducts to the exclusion of higher molecular weight multimer forms suchas trimers and the like. Except for formulations developed specificallyto include non-BPI protein products such as antibiotics and the like asadditional active ingredients, formulations of the invention aresubstantially free of contaminating materials such as DNA, endotoxin andpotentially immunogenic proteins and peptides (including human orrecombinant host cell proteins and peptides) which might disqualify theformulations from regulatory acceptance.

Among the preferred dimeric associations of BPI protein products usedaccording to the invention are those developed by means of recombinantexpression of DNA sequences encoding amino terminal fragments of BPI,i.e., from the initial amino terminal residue (1) to from about residue175 to 199, and thereafter processed in a manner promoting disulfidebond linkages between monomers. Specifically preferred are dimericassociations of the recombinant expression products of DNAs encodingresidues 1 through 199 and residues 1 through 193 of human BPI. Whenexpression takes place in, e.g., Chinese Hamster Ovary cells, thepossibility of development of expression products having variablecarboxy terminal truncations in monomeric forms allows for thegeneration of dimeric forms wherein the termini of the componentmonomers may be unequal in length. The use of dimeric forms of aminoacid addition, substitution and deletion analog BPI protein products isalso contemplated. Analogs involving replacement of a cysteine forexample at positions 132, with an amino acid incapable of forming adisulfide bond (as in the previously-mentioned U.S. Pat. No. 5,420,019the disclosures of which are incorporated by reference herein) is notsusceptible to dimerization without the use of chemical processes, forexample, by means of crosslinking reagents. Analogs wherein cysteinesreplace amino acids, for example at position 18, or are added to aminoor carboxy terminal ends of the BPI protein product will correspondinglybe more susceptible to dimerization.

Also, illustrative of formulations of the invention are those containingBPI holoprotein in the form of a dimetic association of protein monomerswhich are covalently linked by means of disulfide bonds formed betweencysteine amino acid residues extant in the BPI protein. Alsocontemplated are dimeric molecular forms generated through covalentbonding between monomeric forms chemically derivatized by means wellknown in the art.

The present invention also contemplates the therapeutic use of dimericBPI protein products formed by the linkage of immunoglobulin portions ofBPI-immunoglobulin fusion protein variants such as described in thepreviously noted U.S. patent applications Ser. Nos. 07/885,911, nowabandoned, and 08/064,693, now U.S. Pat. No. 5,643,570 the disclosuresof which are incorporated by reference herein. It is further within thecontemplation of the invention that dimeric BPI protein products beprovided as fusion protein products, for example, having a conformationanalogous to that of single chain antibodies, e.g., having a BPI proteinproduct polypeptide at each end of a linear polypeptide, with aminoacids providing a spacer or hinge region permitting the BPI polypeptidesto assume a dimeric conformation.

BPI protein product formulations of the invention provide for improvedtherapeutic treatment of Gram-negative bacterial infection and thesequelae thereof, including exposure to Gram-negative bacterialendotoxin in circulation, bacterial and/or endotoxin-related shock andone or more conditions associated therewith, such as disseminatedintravascular coagulation, anemia, thrombocytopenia, leukopenia, adultrespiratory distress syndrome, renal failure, hypotension, fever, andmetabolic acidosis. Also contemplated are improved therapeutictreatments of mycobacterial infection (by, e.g., M. tuberculosis, M.leprae and M. avium) and the adverse physiological effects oflipoarabinomannan in circulation according to the methods of previouslynoted U.S. patent application Ser. No. 08/031,145, now abandoned, thedisclosure of which is incorporated herein; Helicobacter infectionaccording to the disclosure of U.S. patent application Ser. Nos.08/031,144, now abandoned, and 08/209,479 the disclosures of which areincorporated by reference herein and depressed reticuloendothelial cellsystem function according to the methods of previously noted U.S. patentapplication Ser. No. 08/132,510, now abandoned, the disclosure of whichis incorporated herein.

Improvements of the invention also apply to therapeutic methods for theuse of dimeric BPI protein products as heparin neutralizing agents fortreatment of conditions not directly associated with Gram-negativebacterial infection, including neutralization of the anti-coagulantproperties of heparin, inhibition of angiogenesis, inhibition of tumorcell proliferation, inhibition of endothelial cell proliferation andtreatment of chronic inflammatory disease states such as arthritisaccording to the disclosures of previously noted U.S. patentapplications Ser. Nos. 08/030,644, now U.S. Pat. No. 5,348,942,08/093,202, 08/183,222, both abandoned, and 08/209,762 the disclosuresof which are hereby incorporated by reference.

The invention thus contemplates that administration of BPI proteinproduct dimers will provide beneficial activity for neutralizing theanti-coagulant activity of heparin when administered in vivo to asubject. It is also contemplated that such products will be particularlyuseful when administered to subjects in order to inhibit endothelialcell proliferation and angiogenesis associated with a variety ofconditions including malignant tumor proliferation, Kaposi's sarcomalesions and the like. Cancers susceptible to treatment by administrationof dimeric BPI protein products include melanoma, sarcomas, andcarcinomas including, but not limited to, breast, colon, lung andprostate carcinomas. Other conditions for which dimeric BPI proteinproducts can be administered for inhibition of angiogenesis includeocular retinopathy, retrolental fibroplasia, psoriasis, angiofibromas,endometriosis, hemangiomas and the like. Also contemplated by theinvention are uses of dimeric BPI protein products in methods ofcontraception comprising delivering of an effective mount of a BPIprotein product so as to prevent implantation of a fertilized ovum,methods of treating chronic inflammatory disease states such asarthritis, psoriasis, inflammatory bowel disease, Crohn's disease,ulcerative colitis, lupus erythematosus, autoimmune uveitis, Lymedisease, and asthma.

Antimicrobial therapeutic methods of the present invention may involveadministration of dimeric BPI protein products alone or in combinationwith LBP protein products according to the disclosures of previouslynoted U.S. patent application Ser. No. 08/093,201, now abandoned,incorporated by reference herein, or in combination with antibioticsaccording to the disclosures of previously noted U.S. patent applicationSer. Nos. 08/125,651 and 08/031,145, both abandoned, incorporated byreference herein.

Also provided according to the present invention are novel methods forpreparing compositions comprising high proportions of dimeric forms ofBPI protein products, especially recombinant products, suitable forpharmaceutical use and characterized by being substantially free ofmultimeric BPI protein products, human or host cell proteins orpeptides, polynucleotides and endotoxin. According to one illustrativemethod, recombinant dimeric BPI protein products can be recovered fromrecombinant host cell fermentation by contact one or more times with afirst cationic exchange resin (e.g., SP-Sepharose) and elution therefromunder suitable pH and sodium chloride salt concentrations (e.g., pH 4.0and 1.5M NaCl), followed by contact with a second cationic exchangeresin (e.g., CM-Sepharose), removal of BPI protein product monomer witha 0.5M NaCl solution at pH 4.0 and elution with a 1.0M NaCl solution atpH 4.0 to provide a BPI protein product dimer-containing eluate,followed by contact with and elution from a size exclusion resin (e.g.,Sephacryl S-100) under suitable pH and salt concentration (e.g., pH 5.0and 0.15M NaCl) to provide compositions comprising about 99% pure,dimeric BPI protein product suitable for use in pharmaceuticalformulations.

According to another illustrative method, monomeric BPI protein productcompositions are converted in solution with copper ions, particularlycupric (Cu⁺⁺), ions to provide compositions having high proportions ofdimeric products. Solutions containing BPI protein products insubstantially monomeric form are contacted with cupric ion inappropriate concentrations (e.g., 2 μM to 100 μM), at suitable pH (e.g.,from about pH 5.0 to about pH 9.0), with or without pretreatment withsuitable reducing agents such as dithiothriitol for elimination ofcysteine adducted monomer forms, to provide compositions with 85% ormore dimeric molecular species without reduction or 95% or more withreduction. Use of cupric ion to promote dimer formation is preferablycarried out in the absence of chelating agents such as EDTA or citrateion which tend to inactivate the cupric ion. Applied to recovery ofrecombinant dimeric BPI protein products from host cell fermentation,cupric ion catalyzed conversion of product monomers to dimeric forms isperformed as an intermediate step in a total recovery process which caninclude preliminary steps of contact with and elution from a firstcationic resin (e.g., SP-Sepharose) under pH and salt concentrationsselected to minimize elution of dimeric and multimeric product forms(e.g., pH 4.0 and 1.0M NaCl), contact with and elution from a secondcationic exchange resin (e.g., CM-Sepharose, wash at 0.35M NaCl andelute at 0.60M NaCl) under functionally comparable elution conditionstogether with optional reducing and viral inactivation steps. Recoveryprocedures following cupric ion conversion to dimeric forms can includeapplication to a third cationic exchange resin (e.g., CM-Spherodex)under suitable pH and salt concentration conditions (e.g., pH 5.0 and0.5M NaCl to remove nonconverted monomer and at 1.0M NaCl to elutedimer) followed by contact with and elution from a size exclusion resin(e.g., Sephacryl S-100) under suitable pH and salt concentrationconditions (e.g., pH 5.0 and 0.15M NaCl).

Numerous additional aspects and advantages of the invention will becomeapparent to those skilled in the art upon considering the followingdetailed description of the invention which describes presentlypreferred embodiments thereof, reference being made to the drawingwherein:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the effect of CuSO₄ concentration on conversion of BPImonomer to BPI dimer;

FIG. 2 depicts the conversion of BPI monomer to BPI dimer versus time;

FIG. 3 depicts the effects of BPI protein products on TNF levels in arat LPS infusion endotoxemia model;

FIG. 4 depicts the effects of BPI protein products on mean arterialblood pressure in a rabbit endotoxemia model;

FIG. 5 depicts the effects of BPI protein products on respiratory ratein a rabbit endotoxemia model;

FIG. 6 depicts the effects of BPI protein products on TNF levels in arat LPS infusion endotoxemia model;

FIG. 7 depicts the results of BPI administration in a mouse acuteperitonitis model;

FIG. 8 graphically depicts the ability of BPI protein products to bind¹²⁵ I-LPS while bound to HUVEC cells;

FIG. 9 graphically depicts the ability of BPI protein products to bind¹²⁵ I-LPS while bound to HUVEC cells;

FIG. 10 graphically depicts the results of a heparin binding assay forrBPI₂₃ and rBPI₂₃ dimer;

FIG. 11 graphically depicts the effects of rBPI₂₃ dimer and rBPI₂₃ Δcyson heparin mediated lengthening of thrombin time in vitro;

FIG. 12 depicts the effects of BPI protein products on activated partialthromboplastin time (aPTT) time in heparinized plasma;

FIG. 13 depicts the effects of BPI protein products on activated partialthromboplastin time (aPTT) in heparin-treated rats; and

FIG. 14 graphically depicts pharmacokinetic properties.

DETAILED DESCRIPTION

As used herein, "BPI protein product" includes naturally andrecombinantly produced bactericidal/permeability-increasing protein;natural, synthetic, and recombinant biologically active polypeptidefragments of bactericidal/permeability-increasing protein; andbiologically active polypeptide analogs, including hybrid fusion andvariant proteins, of either bactericidal/permeability increasing proteinor biologically active fragments thereof. BPI protein products includingbiologically active fragments of BPI holoprotein administered accordingto the methods of this invention may be generated and/or isolated by anymeans known in the art. For example, U.S. Pat. No. 5,198,541, thedisclosure of which is hereby incorporated by reference, describesrecombinant methods and materials for expression of BPI proteinsproducts.

The following detailed description relates to the development andproperties of illustrative rBPI protein product formulations accordingto the invention and particularly those comprising dimeric forms ofmonomeric products based on amino terminal fragments of BPI.

In particular, the detailed description illustrates preparative methodsfor the isolation of BPI protein product dimers from heterogeneouspreparations containing monomeric, dimeric and/or other molecular formssuch as cysteine adducted forms and methods for the conversion of BPI inmonomeric or cysteine adduct form into BPI dimers.

Methods for the separation of BPI protein product dimers fromheterogeneous BPI preparations include use of chromatographic separationtechniques. According to one method, the growth medium of transformedmammalian cells producing a BPI protein product is contacted with cationexchange materials such as S-Sepharose or SP-Sepharose. The cationexchange materials are washed with a weak buffer such as 0.65MNaCl-Acetate to remove some impurities and a stronger buffer such as1.0M NaCl-Acetate to elute monomeric products from the exchangematerials, leaving behind dimers and multimers of the BPI proteinproduct on the cation exchange material. Dimers and multimers may thenbe eluted with an even stronger buffer such as 1.5M NaCl-Acetate.Further separations may be practiced to remove contaminants such as DNAfrom the 1.5M NaCl-Acetate eluate and to separate dimeric frommultimeric forms. For example, separation of DNA from BPI proteinproducts can be carried out at pHs of about 4.0.

Dimeric products isolated from fermentation media of cells transformedwith a DNA encoding the BPI signal sequence and the fast 199 residues ofBPI tend to be homogeneous with respect to length of the polypeptidemonomeric subunits involved in formation of the dimers. While monomericproducts isolated from such fermentation media display a variety ofdifferent terminal residues, with the predominant monomeric specieshaving residues 1-193, the dimeric forms isolated directly fromfermentation by chromatographic procedures tend to predominantly includemonomeric subunits having residues 1-199, suggesting that dimerformation in the host cells or the medium "protects" the product fromcarboxy terminal degradation. Conversion of BPI protein product monomersto dimeric forms in vitro by copper sulfate catalyzed dimerization ofisolated monomers leads to recovery of products predominantly includingdimers formed from monomers having residues 1-193.

While BPI protein product monomers will gradually dimerize in solution,such conversion is very slow and is commercially impractical. Theoxidation reaction for rBPI₂₃ proceeds slowly at pH 5 (resulting inapproximately 15% conversion to dimer in one year). The presence ofcuprio (Cu²⁺) ions substantially accelerates conversion of monomer todimer.

The utility of Cu²⁺ ions to efficiently accelerate formation ofbiologically active BPI protein product dimers is surprising in light ofthe disclosures of Engleka et al., J. Biol. Chem. 267: 11307-11315(1992) which notes that copper-oxidized dimerization of human FGF-1proceeds efficiently to generate products which no longer possess eithermitogenic biological activity or heparin binding capacity, but thatsimilar quantitative dimerization by cupric ion oxidation did not occurwhen structurally analogous bovine FGF-1 and recombinant human FGF-2were treated.

The formation of cysteine adducts in a heterogeneous BPI protein productmixture can significantly limit the yield of BPI protein product dimersbecause such cysteine adducts will not dimerize, even in the presence ofcupric ion. In certain cases, the product of a cell culture fermentermay consist of BPI dimer and cysteine adduct with essentially nonon-adducted BPI monomer. According to one aspect of the invention,cysteine adducts in a beterogenous BPI protein product mixture can bereduced to form non-adducted product monomers prior to conversion todimeric form. For example, if BPI is first reduced to eliminate cysteineadducts, about 90% or more of the monomer can be converted by coppersulfate. Conversions can be significantly less over a given time periodwhere cysteine adducts are not so reduced (i.e., about 80% or more in 5hours). Accordingly, a reducing agent such as dithiothreitol (DTT) maybe added to heterogeneous mixtures of dimers, monomers and cysteineadducts in order to produce BPI protein monomer which is then convenedto dimer in high yields. It is contemplated that other reducing agentssuch as Tris(2-carboxyethyl)phosphine (TCEP) will be equally useful inthe reduction of cysteine-adducts.

A variety of factors will influence the overall formation and yield ofdimer. Accordingly, those of skill in the art will know to balance theeffects of product losses with conversion rates in order to maximizeultimate yields. The oxidation of BPI protein product monomer to producedimer is likely a second order reaction with respect to BPIconcentrations. For example, increases in BPI protein productconcentration will increase the dimer formation rate at any givenreaction pH. While the dimer formation rate generally increases withincreasing pH, product yield may be decreased at higher pHs due toprotein insolubility or loss. The dimer formation rate is also dependentupon the concentration of Cu²⁺ ion with concentrations of from about 2μM to about 100 μM Cu²⁺ being preferred. Lower concentrations are lesseffective at promoting the dimerization reaction. The rate ofdimerization is also a function of temperature. Specifically, dimerformation occurs more rapidly at room temperature than at 4° C. althoughprotein stability may be greater at the reduced temperature. Bufferselection may also influence dimer formation. However, experimentscomparing Tris and phosphate buffers found no significant differencebetween the two with respect to rate of dimer formation or the ultimatepercentage of dimer recovered. On the other hand, the incorporation ofcitrate buffer has the effect of chelating the Cu²⁺ ion and willsubstantially reduce the rate of dimerization. Similarly, highconcentrations of phosphate may have the effect of precipitating cupricion. Variations in NaCl concentrations ranging from 0.1 to 1.7M NaCl didnot substantially affect dimer yields.

As one aspect of the invention it has been found that cationic exchangeresins such as those used in the purification procedures of theinvention or in the improved cell culture methods of co-owned andcopending U.S. Pat. No. 5,439,807 may promote BPI protein product dimerformation by means of physical or charge interactions. Accordingly,dimer formation can be efficiently carried out in the cell culturefermenter and/or on the media of cationic exchange purification columns.To the extent that it is desired to promote dimer formation in the cellculture fermenter it may be desirable to alter the concentration ofcationic exchange resin in the fermenter in order to influence dimerformation. In addition, the cupric ion concentration of the cell culturefermentation media can be increased in order to promote dimer formationin that media. Further, it may also be desirable to reduce the cysteineor cystine concentration of the fermentation media in order to repressformation of cysteine adducts. Nevertheless, such cysteineconcentrations should not be so reduced as to be rate limiting for cellgrowth.

The administration of BPI dimers is preferably accomplished with apharmaceutical composition comprising the product and a pharmaceuticallyacceptable diluent, adjuvant or carrier. The BPI protein productcomposition may be administered without or in conjunction with knownantibiotics, surfactants or other chemotherapeutic agents. A preferredpharmaceutical composition comprises the BPI protein at a concentrationof 1 mg/ml in citrate buffered saline (0.02M citrate, 0.15M NaCl, pH5.0) comprising 0.1% by weight of poloxamer 188 (Pluronic F-68, BASFWyandotte, Parsippany, N.J.) and 0.002% by weight of polysorbate 80(Tween 80, ICI Americas Inc., Wilmington, Del.). Such preferredcombinations are taught in co-owned, copending U.S. patent applicationSer. No. 08/012,360 filed Feb. 2, 1993, now abandoned, andcontinuation-in-part Ser. No. 08/190,869 filed Feb. 2, 1994, now U.S.Pat. No. 5,448,034, the disclosures of which are herein incorporated byreference.

The BPI protein product can be administered by any known method, such asorally, systemically (such as by intravenous, intramuscular or otherinjection), or as an aerosol. Medicaments can be prepared for oraladministration or by injection or other parenteral methods andpreferably include conventional pharmaceutically acceptable carriers andadjuvents as would be known to those of skill in the art. Themedicaments may be in the form of a unit dose in solid, semi-solid andliquid dosage forms such as tablets, pills, powders, liquid solutions orsuspensions, and injectable and infusible solutions. Effective dosageranges from about 100 μg/kg to about 100 mg/kg of body weight arecontemplated.

The present invention will be better understood upon consideration ofthe following illustrative examples wherein: Example 1 relates toconstruction of vectors for expression of BPI protein products; Example2 addresses the incorporation of vectors of Example 1 into appropriatehost cells for the expression of preferred BPI protein productpolypeptides; Example 3 relates to methods suitable for the isolation ofdimeric products from the medium of CHO cells transfected with DNAencoding the secretory signal and BPI residues 1-199; Example 4 relatesto evaluation of various divalent metal ions for promotion of dimerformation; Example 5 relates to methods for the conversion to dimericforms; Example 6 relates to methods for the purification and conversionto dimeric products; Example 7 describes evaluation of BPI proteinproducts including dimer-containing formulations in a rat LPS infusion(experimental endotoxemia) model; Example 8 relates to evaluation of BPIprotein products including dimer-containing formulations in a rabbitendotoxemia model; Example 9 relates to the activity of monomeric BPIprotein products in a rat LPS infusion model; Example 10 relates to theefficacy of BPI protein product dimer-containing formulations in a mouseacute peritonitis model; Example 11 relates to the effect of BPI dimerin a mouse endotoxemia model; Example 12 relates to the pharmacokineticsof dimer-containing formulations in rats; Example 13 relates to theability of BPI protein product formulations including dimeric molecularforms prebound to human umbilical vein endothelial (HUVEC) cells to bindLPS; Example 14 relates to quantification of heparin binding by BPIdimer; Example 15 related to the effect of BPI dimer on heparin-mediatedlengthening of thrombin time; Example 16 relates to the effect ofdimer-containing formulations on heparin-mediated lengthening of partialthromboplastin time in vitro and in vivo; and Example 17 relates to theeffect of dimer-containing formulations on heparin neutralization in aMatrigel™ model of angiogenesis.

EXAMPLE 1 Construction of Vectors for Expression of rBPI₂₁ Δcys andOptimized Vectors for Expression of rBPI (1-193)

In this example, construction of vectors for expression of a variety ofBPI protein products including the product of expression of a DNAencoding residues 1-193 of BPI wherein the cysteine at position 132 isreplaced by alanine (referred to herein as rBPI₂₁ Δcys) and the productof expression of a DNA encoding residues 1-193 of BPI (referred toherein as rBPI (1-193) and the product of expression of DNA encodingresidues 1-199 of BPI (referred to herein as rBPI₂₃) are disclosed. TherBPI (1-193)product is a presently preferred source of monomer forpreparation of dimeric BPI protein products for practice of theinvention.

A. Construction of Plasmids pING4519 and pING4520

The expression vector, pING4503, was used as a source of DNA encoding arecombinant expression product designated rBPI (1-199), i.e., encoding apolypeptide having the 31-residue signal sequence and the first 199amino acids of the N-terminus of the mature human BPI, as set out in SEQID NOs: 1 and 2 except that valine at position 151 is specified by GTGrather than GTC and residue 185 is glutamic acid (specified by GAG)rather than lysine (specified by AAG).

Plasmid pING4503 has been described in co-pending, co-owned U.S. patentapplication Ser. No. 07/885,911 by Theofan, et al., now abandoned, whichis incorporated herein by reference with respect to the background ofthe invention. Briefly, the construction of pING4503 is based on plasmidpING2237N which contains the mouse immunoglobulin heavy chain enhancerelement, the LTR enhancer-promoter element from Abelson murine leukemiavirus (A-MuLv) DNA, the SV40 19S/16S splice junction at the 5' end ofthe gene to be expressed, and the human genomic gamma-1 polyadenylationsite at the 3' end of the gene to be expressed. Plasmid pING2237N alsohas a mouse dihydrofolate reductase (DHFR) selectable marker. The DNAencoding rBPI (1-199), including 30 bp of the natural 5' untranslatedregion and bases encoding the 31 amino acid signal sequence, as well as199 N-terminal amino acids of BPI, is inserted between unique SalI andSstII restriction sites in pING4503.

Two vectors, pING4519 and pING4520, were constructed based on pING4503for expression of rBPI (1-199) cysteine replacement analogs in which oneof the three naturally-occurring cysteine residues of BPI was replacedwith another amino acid. A PvuII site (CAGCTG) which occurs only once inthe DNA encoding rBPI (1-199), and which is located between cysteine 132and cysteine 135, was utilized in these constructions. Because severaladditional PvuII sites exist in pING4503, it was first necessary toisolate the SalI-SstII fragment which contained the insert encoding rBPI(1-199) from pING4503 by digesting with SalI and SstII. The purifiedSalI-SstII rBPI (1-199) insert was then digested with PvuII, resultingin an approximately 529 bp SalI-PvuII fragment and an approximately 209bp PvuII-SstII fragment, each of which was purified separately.

Plasmid pING4519 is identical to pING4503 except that pING4519 containsa DNA insert encoding an rBPI (1-199) in which a codon for alanine issubstituted for the codon specifying the native cysteine at position132. The recombinant product resulting from host cell expression andrecretory processing of such an insert is referred to as "rBPI (1-199)ala¹³²." In order to generate pING4519, BPI DNA sequences were PCRamplified from pING4503 using the primers BPI-6:AAGCTTGTCGACCAGGCCTTGAGGT (SEQ ID NO: 3), which incorporated a SalIrestriction site at the 5' end of the 30 bp BPI untranslated region, andBPI-14: CTGGAGGCGGTGATGGTG (SEQ ID NO: 4), which incorporated one halfof the PvuII site and the base substitutions necessary to code foralanine at position 132. PCR amplification was accomplished using theGeneAmp PCR kit (Perkin Elmer Ceres, Norwalk, Conn.) according to themanufacturer's instructions. The resulting PCR fragment was digestedwith SalI, resulting in an approximately 529 bp SalI-blunt fragmentwhich was then used in a three-piece ligation, together with theapproximately 209 bp PvuII-SstII fragment described above and the largefragment resulting from SalI and SstII digestion of pING4503, togenerate pING4519.

Plasmid pING4520 is identical to pING4519 with the exception thatpING4520 contains a DNA insert encoding an rBPI (1-199) analog in whicha serine codon is substituted for the codon specifying the nativecysteine at position 135. The recombinant product resulting from hostcell expression of such an insert is designated "rBPI (1-199) ser¹³⁵ ".In order to generate pING4520, BPI DNA sequences were PCR amplified frompING4513, a plasmid essentially similar to pING4503 except that theselection marker is gpt instead of DHFR and the cDNA insert encodes thesignal sequence and full-length BPI (456 residues) instead of only therBPI (1-199) portion.

Amplification by PCR was accomplished using primer BPI-15:CTCCAGCAGCCACATCAAC (SEQ ID NO: 5), wherein the 5' end incorporates onehalf of a mutated PvuII site (wherein "CTG" is changed to "CTC") and thebase substitutions necessary to code for refine at position 135; andprimer BPI-7: GAACTTGGTTGTCAGTCG (SEQ ID NO: 6), representingrBPI-encoding sequences located downstream of the region encoding BPIresidue 199. This PCR fragment was digested with BstBI, which cutsdownstream of the cysteine 135 mutagenesis site, and the resultingapproximately 100 bp blunt-BstBI fragment was gel purified. A threepiece ligation was then performed with the 529 bp SalI-PvulI BPIrestriction fragment described above, the 100 bp blunt-BstBI fragment,and a large fragment resulting from BstBI-SalI digestion of pING4503, togenerate pING4520.

B. Construction of Plasmid pING4530

Another vector, pING4530, was constructed which contained thealanine-for-cysteine replacement as in pING4519, but which contained thegpt selectable marker (allowing for mycophenolic acid resistance)instead of the DHFR marker carried over from pING4503 to pING4519. Toconstruct pING4530, a 1629 bp SalI-DraIII restriction fragment wasisolated from pING4519. This fragment included all of the rBPI (1-199)ala¹³² coding region as well as an additional approximately 895 bpvector sequence at the 3' end of the coding region. This fragment wasligated to the large (approximately 7230 bp) DraIII-SalI vector fragmentisolated from pING4513 to generate pING4530.

C. Construction of Plasmid pING4533

Plasmid pING4533 was constructed for expression of rBPI (1-199) ala¹³²,wherein the codon specifying the fifth amino acid of the BPI signalsequence, methionine (ATG), at position -27 was placed in the context ofthe consensus Kozak translation initiation sequence GCCACCRCCATGG (SEQID NO: 7) Kozak, Nucl. Acid. Res., 15:8125 (1987)!, and in which the DNAsequence encoding the first 4 amino acids of the BPI signal was removed.This was accomplished by PCR amplification of BPI sequences from aplasmid containing the full length human BPI cDNA in pGEM-7zf(+)! usingthe PCR primer BPI-23: ACTGTCGACGCCACCATGGCCAGGGGC (SEQ ID NO: 8),incorporating a SalI restriction site and the nucleotides GCCACC infront of the ATG (methionine) at position -27 of the BPI signal, and theprimer BPI-2: CCGCGGCTCGAGCTATATTTTGGTCAT (SEQ ID NO: 9), correspondingto the 3' end of the rBPI (1-199) coding sequence.

The approximately 700 bp PCR amplified DNA was digested with SalI andEcoRI and the resulting 270 bp fragment, including approximately thefirst one-third of the BPI (1-199) coding sequence, was purified. ThisSalI-EcoRI fragment was ligated to 2 other fragments: (1) a 420 bpEcoRI-SstII fragment from pING4519, encoding the remainder of BPI(1-199) wherein alanine replaces cysteine at position 132; and (2) anapproximately 8000 bp SstII-SalI vector fragment from pING4502 (a vectoressentially similar to pING4503 except that it does not include the 30bp 5' untranslated sequence and has a gpt marker rather than DHFR), togenerate pING4533 which contains a gpt marker.

D. Construction of Plasmids pING4221, pING4222, and pING4223

Vectors similar to pING4533 were constructed having an insert whichcontained the optimized Kozak translation initiation site correspondingto methionyl residue -27 of the signal sequence, and analanine-for-cysteine replacement at position 132. However, the BPIfragment coding sequence terminated at residue 193 in theseconstructions. The recombinant product resulting from host cellexpression of this DNA is referred to as "rBPI (1-193) ala¹³² " or as"rBPI₂₁ Δcys." Vectors containing these inserts were made by firstdigesting pING4533 with SalI, which cuts at the 5' end of the BPI DNAinsert, and AlwNI, which leaves a three bp 3'-overhang at residue 192.The resulting approximately 700 bp fragment was then purified. Thisfragment was re-ligated into the large fragment resulting from pING4533digestion with SstII-SalI, along with two annealed complementaryoligonucleotides, BPI-30: CTGTAGCTCGAGCCGC (SEQ ID NO: 10) and BPI-31:GGCTCGAGCTACAGAGT (SEQ ID NO: 11). This replaced the region between theAlwNI and SstII sites with the codon for residue 193 (leucine), a stopcodon, and an XhoI restriction site 5' to the SstII site and resulted inregeneration of both the AlwNI and the SstII sites and placement of thestop codon, TAG, immediately after the codon (CTG) for amino acid 193(leucine). The resultant plasmid was designated pING4223 and had the gptmarker. Similar constructions were made exactly as described forpING4223 except that different SstII-SalI vector fragments were used togenerate vectors with different selection markers. For example, pING4221is identical to pING4223 except that it contains the his marker(conferring resistance to histidinol) instead of gpt and pING4222 isidentical to pING4223 except that it contains the DHFR marker instead ofgpt.

E. Construction of Plasmids pING4537, pING4143, pING4146, pING4150, andpING4154

A series of vectors was constructed which contained an insert encodingrBPI (1-193) ala¹³², the optimized Kozak transhtion initiation site, anddifferent selection markers essentially identical to those describedwith respect to pING4221, pING4222 and pING4223 except that the humangenomic gamma-1 heavy chain polyadenylation and transcriptiontermination region at the 3' end of the SstII site was replaced with ahuman light chain polyadenylation sequence followed by mouse light chain(kappa) genomic transcription termination sequences. In collateral geneexpression studies, the light chain polyadenylation signal andtranscription termination region appeared to be responsible for 2.5-5fold increases in BPI expression levels in Sp2/0 and CHO-K1 cells.

The aforementioned vectors were constructed by first constructingpING4537, a vector similar to pING4533 which contains the rBPI (1-199)ala¹³² insert. However, pING4537 includes the human light chainpolyadenylation and mouse kappa genomic transcription terminationsequences instead of the human heavy chain sequence. These 3' sequenceswere obtained from pING3170, an expression vector which encodes a humanlight chain cDNA and includes a mouse genomic light chain 3'transcription termination sequence. This was accomplished by digestingwith SstI, which cuts 35 bp upstream of the human stop codon, treatingwith T4 DNA polymerase to make the end blunt, then cutting with BamHI,and purifying an approximately 1350 bp fragment which includes the mousekappa 3' sequences. The resulting fragment consists of approximately 250bp of the 3' portion of the human light chain constant region cDNA andthe polyadenylation signal followed by a BamHI linker as described inthe construct called Δ8 in Lui et al.,J. Immunol 139: 3521, (1987). Theremainder of the approximately 1350 bp fragment consists of aBglII-BamHI mouse kappa 3' genomic fragment fragment "D" of Xu et al.,J. Biol. Chem. 261:3838, (1986)! which supplies transcriptiontermination sequences. This fragment was used in a 3-piece ligation withtwo fragments from pING4533: a 3044 bp fragment which includes all ofBPI insert and part of vector obtained by digestion with SstII, T4polymerase treatment, and NotI digestion (which includes all of BPIinsert and part of vector), and an approximately 4574 bp BamHI-NotIfragment. The resulting vector, pING4537, is identical to pING4533 withthe exception of the above-noted differences in the genomic 3'untranslated region.

Additional vectors containing the kappa 3' untranslated sequences wereconstructed using pING4537 as the source of the kappa 3' fragment. Thekappa 3' untranslated sequences were isolated by digestion of pING4537with XhoI (a unique site which occurs immediately after the BPI stopcodon) and BamHI. The resulting approximately 1360 bp XhoI-BamHIfragment was used in a series of 3-piece ligations to generate thefollowing four vectors, all of which have inserts encoding rBPI (1-193)ala¹³² and which have the optimized Kozak translation initiation site atresidue -27 of the signal: (1) pING4143 (gpt marker), obtained byligating a pING4223 4574 bp BamHI-NotI fragment (gpt marker), a pING4223NotI-XhoI BPI insert-containing fragment of approximately 3019 bp, andthe pING4537 XhoI-BamHI fragment; (2) pING4146 (DHFR marker), obtainedby ligating a pING4222 approximately 4159 bp BamHI-NotI fragment (DHFRmarker), a pING4223 NotI-XhoI BPI insert-containing fragment ofapproximately 3019 bp, and the pING4537 XhoI-BamHI fragment; (3)pING4150 (his marker), obtained by ligating a pING4221 his-containingapproximately 4772 bp BamHI-NotI fragment, a pING4222 NotI-XhoI BPIinsert-containing fragment, and the pING4537 XhoI-BamHI fragment; and(4) pING4154 (neo marker), obtained by ligating a pING3174neo-containing approximately 4042 bp BamHI-BsaI fragment, a pING4221BsaI-XhoI BPI insert-containing fragment of approximately 3883 bp andthe pING4537 XhoI-BamHI fragment. Plasmid pING3174 contains an insertencoding antibody heavy chain DNA and has a neo marker. The neo gene andits flanking sequences were obtained from the pSv2 neo plasmid reportedby Southern et al., J. Mol. Appl. Genet., 1:327 (1982).

F. Construction of Plasmids pING4144 And pING4151

Two plasmids were constructed, pING4144 and pING4151, which wereidentical to pING4143 and pING4150, respectively, except that expressionof rBPI coding sequences was under control of the human cytomegalovirus(hCMV) immediate early enhancer/promoter instead of the Abelson murineleukemia virus (A-MuLv) LTR promoter. Therefore, both pING4144 andpING4151 contained the mutation of the cysteine at position 132 toalanine, the optimized Kozak translation initiation sequence, and thehuman light chain poly-A/mouse kappa genomic transcription terminationregion. The region between nucleotides 879 and 1708 of the originalvectors (pING4143 and pING4150) was replaced with a region of the hCMVenhancer/promoter corresponding to nucleotides -598 through +174 asshown in FIG. 3 of Boshart et al., Cell 41:521 (1985), incorporatedherein by reference. To introduce the hCMV promoter region into BPIexpression vectors, plasmid pING4538 was first constructed by replacingthe approximately 1117 bp EcoRI-SalI/A-MuLv promoter-containing fragmentof pING4222 with an approximately 1054 bp EcoRI-SalI/hCMVpromoter-containing fragment from plasmid pING2250 which contains thehCMV promoter driving expression of an antibody light chain insert. Toconstruct pING4144, three fragments were ligated together: (1) theapproximately 2955 bp rBPI (1-193)-containing NotI-XhoI fragment frompING4538; (2) the approximately 1360 bp XhoI-BamHI fragment frompING4537; and (3) the approximately 4770 bp BamHI-NotI fragmentcontaining the his gene from pING4221.

G. Construction of Plasmids pING4145, pING4148 and pING4152

Plasmids pING4145, pING4148 and pING4152 were constructed and wereidentical to pING4143, pING4146, and pING4150, respectively, except thatthey contained the natural sequence cysteine at position 132 instead ofan alanine substitution. Thus, all three contained the rBPI (1-193)insert, the optimized Kozak translation initiation sequence and thehuman light chain Poly A/mouse kappa genomic transcription terminationregion. These three plasmids were constructed as follows. To constructpING4145, three fragments were ligated together: (1) the approximately3000 bp NotI-XhoI BPI (1-193) containing fragment from pING4140(pING4140 is identical to pING4221 except that it contains the naturalsequence cysteine at Position 132); (2) the approximately 1360 bpXhoI-BamHI fragment from pING4537; and (3) the approximately 4570 bpBamHI-NotI fragment containing the gpt gene from pING4223. To constructpING4148, three fragments were ligated together: (1) the NotI-XhoIfragment from pING4140; (2) the XhoI-BamHI fragment from pING4537; and(3) the approximately 4150 bp BamHI-NotI fragment containing the DHFRgene from pING4222. To construct pING4152, three fragments were ligatedtogether: (1) the approximately 3000 bp NotI-XhoI fragment from pING4142(pING4142 is identical to pING4223 except that it contains the naturalsequence cysteine at 132); (2) the XhoI-BamHI fragment from pING4537;and (3) the approximately 4770 bp BamHI-NotI fragment containing the hisgene from pING4221.

H. Construction of Plasmids pING4147, pING4153, pING4149 and pING4063

Four plasmids incorporating different selection markers were constructedfor expression of BPI (1-193) in the optimized expression vectors. Thesevectors all included the CMV promoter, the optimized Kozak translationinitiation sequence at position -27 of the signal peptide, and the humankappa poly A/mouse kappa genomic transcription termination sequences.These four plasmids, whose construction are described below, are:pING4147 (gpt), pING4153 (his), pING4149 (DHFR) and pING4063 (neo).

pING4147, containing the gpt marker, is identical to pING4145 (Table I)except that it contains the CMV promoter instead of the A-MuLV promoter.pING4147 was constructed by ligating three fragments together: the ˜6000bp BstBI-NotI vector fragment of pING4144 (Table I) containing the lightchain 3' sequences and the gpt marker, the ˜1900 bp NotI-SalI fragmentof pING4144 containing the CMV promoter region, and the ˜600 bpSalI-BstBI BPI-containing fragment of pING4142. pING4142 is identical topING4145 (Table I) except that it contains the human genomic heavy 3'sequences instead of the light chain 3' sequences.

pING4153, which contains the his marker, was constructed by ligatingthree fragments together: the ˜4000 bp BamHI-NotI fragment of pING4221(Table I) containing the his marker, the ˜2700 bp NotI-XhoI fragment ofpING4147 (described above) containing the CMV promoter and BPI insertsequences, and the ˜1300 bp XhoI-BamHI fragment of pING4537 (describedin section 1E) containing the light chain 3' sequences.

pING4149, which contains the DHFR gene for selection, was constructed byligating these two fragments together: the ˜5900 bp XhoI-NotI vectorfragment of pING4540 including the DHFR gene, and the ˜2700 bp NotI-XtoIfragment of pING4147 (described above) containing the CMV promoter andBPI insert sequences. pING4540 is identical to pING4144 (Table I) exceptthat it contains the DHFR gene instead of gpt for selection.

pING4063 is identical to pING4147 (described above) except that itcontains the neo selection marker instead of the gpt. pING4063 wasconstructed by ligating the ˜2900 bp NotI-XhoI fragment from pING4149(described above) with the XhoI-NotI vector fragment from pING4154(Table I), supplying the light chain 3' sequences and the neo selectionmarker.

Table I, below, summarizes the content of the plasmids whose preparationis described in Sections A through H above.

                  TABLE I                                                         ______________________________________                                                          Signal         3'                                           Plasmid                                                                              BPI Product                                                                              Seq.     Marker                                                                              Terminal                                                                             Promoter                              ______________________________________                                        pING4519                                                                             (1-199)Ala.sup.132                                                                       31AA     DHFR* Human  A-MuLv                                                                 Genomic                                                                       HC                                                                            Gamma-1                                                                       Poly-A                                       pING4520                                                                             (1-199)Ser.sup.135                                                                       31AA     DHFR* Human  A-MuLv                                                                 Genomic                                                                       HC                                                                            Gamma-1                                                                       Poly-A                                       pING4530                                                                             (1-199)Ala.sup.132                                                                       31AA     gpt   Human  A-MuLv                                                                 Genomic                                                                       HC                                                                            Gamma-1                                                                       Poly-A                                       pING4533                                                                             (1-199)Ala.sup.132                                                                       Kozak    gpt   Human  A-MuLv                                                  initiation     Genomic                                                        Seq;           HC                                                             27AA           Gamma-1                                                        signal         Poly-A                                       pING4223                                                                             (1-193)Ala.sup.132                                                                       Kozak    gpt   Human  A-MuLv                                                  initiation     Genomic                                                        Seq;           HC                                                             27AA           Gamma-1                                                        signal         Poly-A                                       pING4221                                                                             (1-193)Ala.sup.132                                                                       Kozak    his   Human  A-MuLv                                                  initiation     Genomic                                                        Seq;           HC                                                             27AA           Gamma-1                                                        signal         Poly-A                                       pING4222                                                                             (1-193)Ala.sup.132                                                                       Kozak    DHFR  Human  A-MuLv                                                  initiation     Genomic                                                        Seq;           HC                                                             27AA           Gamma-1                                                        signal         Poly-A                                       pING4537                                                                             (1-199)Ala.sup.132                                                                       Kozak    gpt   Human  A-MuLv                                                  initiation     Kappa                                                          Seq;           Poly-A/                                                        27AA           Mouse                                                          signal         Kappa                                                                         Genomic                                                                       Transcrip-                                                                    tion                                                                          Termina-                                                                      tion                                         pING4143                                                                             (1-193)Ala.sup.132                                                                       Kozak    gpt   Human  A-MuLv                                                  initiation     Kappa                                                          Seq;           Poly-A/                                                        27AA           Mouse                                                          signal         Kappa                                                                         Genomic                                                                       Transcrip-                                                                    tion                                                                          Termina-                                                                      tion                                         pING4146                                                                             (1-193)Ala.sup.132                                                                       Kozak    DHFR  Human  A-MuLv                                                  initiation     Kappa                                                          Seq;           Poly-A/                                                        27AA           Mouse                                                          signal         Kappa                                                                         Genomic                                                                       Transcrip-                                                                    tion                                                                          Termina-                                                                      tion                                         pING4150                                                                             (1-193)Ala.sup.132                                                                       Kozak    his   Human  A-MuLv                                                  initiation     Kappa                                                          Seq;           Poly-A/                                                        27AA           Mouse                                                          signal         Kappa                                                                         Genomic                                                                       Transcrip-                                                                    tion                                                                          Termina-                                                                      tion                                         pING4144                                                                             (1-193)Ala.sup.132                                                                       Kozak    gpt   Human  hCMV                                                    initiation     Kappa                                                          Seq;           Poly-A/                                                        27AA           Mouse                                                          signal         Kappa                                                                         Genomic                                                                       Transcrip-                                                                    tion                                                                          Termina-                                                                      tion                                         pING4145                                                                             (1-193)    Kozak    gpt   Human  A-MuLv                                                  initiation     Kappa                                                          Seq;           Poly-A/                                                        27AA           Mouse                                                          signal         Kappa                                                                         Genomic                                                                       Transcrip-                                                                    tion                                                                          Termina-                                                                      tion                                         pING4148                                                                             (1-193)    Kozak    DHFR  Human  A-MuLv                                                  initiation     Kappa                                                          Seq;           Poly-A/                                                        27AA           Mouse                                                          signal         Kappa                                                                         Genomic                                                                       Transcrip-                                                                    tion                                                                          Termina-                                                                      tion                                         pING4152                                                                             (1-193)    Kozak    his   Human  A-MuLv                                                  initiation     Kappa                                                          Seq;           Poly-A/                                                        27AA           Mouse                                                          signal         Kappa                                                                         Genomic                                                                       Transcrip-                                                                    tion                                                                          Termina-                                                                      tion                                         pING4151                                                                             (1-193)Ala.sup.132                                                                       Kozak    his   Human  hCMV                                                    initiation     Kappa                                                          Seq;           Poly-A/                                                        27AA           Mouse                                                          signal         Kappa                                                                         Genomic                                                                       Transcrip-                                                                    tion                                                                          Termina-                                                                      tion                                         pING4154                                                                             (1-193)ala.sup.132                                                                       Kozak    neo   Human  A-MuLv                                                  initiation     Kappa                                                          Seq;           Poly-A/                                                        27AA           Mouse                                                          signal         Kappa                                                                         Genomic                                                                       Transcrip-                                                                    tion                                                                          Termina-                                                                      tion                                         pING4147                                                                             (1-193)    Kozak    gpt   Human  CMV                                                     initiation     Kappa                                                          Seq;           Poly-A/                                                        27AA           Mouse                                                          signal         Kappa                                                                         Genomic                                                                       Transcrip-                                                                    tion                                                                          Termina-                                                                      tion                                         pING4149                                                                             (1-193)    Kozak    DHFR  Human  CMV                                                     initiation     Kappa                                                          Seq;           Poly-A/                                                        27AA           Mouse                                                          signal         Kappa                                                                         Genomic                                                                       Transcrip-                                                                    tion                                                                          Termina-                                                                      tion                                         pING4153                                                                             (1-193)    Kozak    his   Human  CMV                                                     initiation     Kappa                                                          Seq;           Poly-A/                                                        27AA           Mouse                                                          signal         Kappa                                                                         Genomic                                                                       Transcrip-                                                                    tion                                                                          Termina-                                                                      tion                                         pING4063                                                                             (1-193)    Kozak    neo   Human  CMV                                                     initiation     Kappa                                                          Seq;           Poly-A/                                                        27AA           Mouse                                                          signal         Kappa                                                                         Genomic                                                                       Transcrip-                                                                    tion                                                                          Termina-                                                                      tion                                         ______________________________________                                         *An altered DHFR gene as described in copending, coowned U.S. patent          application Ser. No. 07/885,911 now abandoned, incorporated herein by         reference.                                                               

EXAMPLE 2 Stable Transfection of Mammalian Cells for Expression of BPIProtein Products

Mammalian cells arc preferred hosts for production of rBPI proteinproducts according to the invention because such cells allow for propersecretion, folding, and post-translational modification of expressedproteins. Presently preferred mammalian host cells for production ofproducts of the invention include cells of fibroblast and lymphoidorigin, such as: CHO-K1 cells (ATCC CCL61); CHO-DG44 cells, adihydrofolate reductase deficient DHFR⁻ ! mutant of CHO Toronto obtainedfrom Dr. Lawrence Chasin, Columbia University; CHO-DXB-11, a DHFR mutantof CHO-K1 obtained from Dr. Lawrence Chasin; Vcro cells (ATCC CRL81);Baby Hamster Kidney (BHK) cells (ATCC CCL10); Sp2/O-Ag14 hybridoma cells(ATCC CRL1581); and NSO myeloma (ECACC No. 85110503).

Transfecfion of mammalian cells may be accomplished by a variety ofmethods. A common approach involves calcium phosphate precipitation ofexpression vector DNA which is subsequently taken up by host cells.Another common approach, electroporation, causes cells to take up DNAthrough membrane pores created by the generation of a strong electricfield (Sambrook et al., Molecular Cloning, A Laboratory Manual, ColdSpring Laboratory Harbor Press, 16.30-16.31 (1989)!. Selection fortransfected cells is facilitated by incorporation in the expressionvector of a gene whose product allows the transfected cells to surviveand grow under selective conditions. A number of such genes have beenidentified. These include, among others: (1) the bacterial Tn5 neo gene,a prokaryotic gene which encodes resistance to the aminoglycosideantibiotic G418; (2) E. coli guanine phosphoribosyl transferase (gpt),which encodes resistance to mycophenolic acid (MPA) in the presence ofxanthine, Mulligan et al., Proc. Nat. Acad. Sci. USA, 78:2072-2076(1981)!; (3) dihydrofolate reductase (DHFR), which allows for growth ofDHFR⁻ cells in the absence of nucleosides and gene amplification in thepresence of increasing concentration of methotrexate; (4) the hisD geneof Salmonella typhimurium which allows growth in the presence ofhistidinol Hartman et al., Proc. Nat. Acad. Sci. USA, 85:8047-8051,(1988)!; (5) the trpB gene of E. coli Hartman et al., Proc. Nat. Acad.Sci. USA, 85:8047-8051, (1988)!, which allows growth in the presence ofindole (without tryptophan); and (6) the glutamine synthetase gene,which allows growth in media lacking glutamine and gene amplification inthe presence of methionine sulfoximine. The availability of theseselective markers, either alone or in various combinations, providesflexibility in the generatiim of mammalian cell lines which expressrecombinant products at high levels.

A. Transfection of pING4145 into CHO-K1 Cells

Plasmid pING4145 contains gene sequences encoding rBPI (1-193) fused tothe A-MuLv promoter, the optimized Kozak translation initiationsequence, the mouse kappa light chain 3' untranslated sequences, alongwith the gpt marker for selection of MPA-resistant cells.

The CHO-K1 cell line was transfected with pING4145 DNA using the calciumphosphate method. Following transfection, the cells were allowed torecover for 24 hours in non-selective Ham's F12 medium. The cells werethen trypsimized, resuspended at a concentration of about 2.5 and 5×10⁴cells/ml in Ham's F12 medium supplemented with MPA (25 μg/ml) andxanthine (250 μg/ml) and then plated at about 5×10³ and 10⁴ cells/wellin 96-well plates. Untransfected CHO-K1 cells are unable to grow in thismedium due to the inhibition of pyrimidine synthesis by MPA.

At approximately 2 weeks, supernatants from 125 wells containing singlecolonies were analyzed for the presence of BPI-reactive protein byanti-BPI ELISA. In this assay, Immulon-II96-well plates (Dynatech,Chantilly, Va.) were pre-coated with affinity purified rabbit anti-rBPI(1-199) antiserum. Supernatant samples were added and detection wascarried out, using affinity purified, biotinylated rabbit anti-rBPI(1-199) antiserum and peroxidase labelled avidin.

The top producers were transferred to 24-well plates for productivityassessment. Cells were grown to confluence in a 24-well plate in Ham'sF12 medium supplemented with 10% FBS. Once the cells reached confluence,the Ham's F12 medium was removed and 1 ml of HB-CHO serum free medium(Irvine Scientific) plus 40 μl of sterile S-sepharose. beads (Pharmacia,Piscataway, N.J.) was added as in co-owned, copending U.S. Pat. No.5,439,807 by Grinna. The cells were then incubated for 7 days afterwhich the S-sepharose beads were removed and washed with 0.1M NaCl in 10mM Tris buffer (pH 7.5). The product was eluted from the beads byaddition of 1.0M NaCl in Tris buffer and quantitated by ELISA asdescribed above. The top-producing transformant, designated Clone C16was chosen for re-transfection with a second plasmid, pING4063 toprovide a cell line which produces optimal levels of BPI.

B. Transfection of pING4063 into CHO-K1/pING4145/Clone C16

Plasmid pING4063 is similar to pING4145 except that it contains thehuman cytomegalovirus (hCMV) promoter instead of the A-MuLv promoter,and the neo gene for selection of G418-resistant cells.

The CHO-K1 Clone C16 cell line was transfected with pING4063 DNA byelectroporation. Following a 48 hour recovery in Ham's F12 medium, thecells were plated in selective medium (MPA plus xanthine, as above, plus0.6 mg/ml G418) in the manner described above in section A. Atapproximately two weeks, supernatants from 252 wells containing singlecolonies were screened for the presence of BPI-reactive protein byELISA. The top producers were transferred to 24 well plates and BPIexpression determined in 24 well plates containing S-sepharose asdescribed above in section A. The top producer, Clone C212, was adaptedto Excell 301 serum-free medium (JRH Scientific, Lenexa, Kans.),containing the selective agents as above in preparation for growth infermenters. The adapted cells were grown in 1.5 L fermenters containingExcell 301 medium supplemented with 2% fetal bovine serum. Productivitywas assessed at 200-260 hours. The productivity was approximately 20-30mg/L at this stage of the fermentation. This done, re-designated cloneC2068, was deposited with the American Type Culture Collection, 12301Parklawn Drive, Rockville, Md. 20852 as ATCC accession No. CRL 11535.

EXAMPLE 3 Isolation of BPI Dimer Products Prom Cell Culture Medium

In this example substantially pure dimeric product was isolated awayfrom monomer products, multimer products and other materials such asnucleic acids present in the medium of transfected CHO host cells.Specifically, CHO host cells transfected with DNA encoding the secretorysequence and residues 1-199 of BPI were grown in a 750 L fermenter inthe presence of a growth medium consisting of Ex-Cell 301 (J.R.H.Corporation, Lenexa, Kans.) supplemented with 0.5% fetal bovine serum,xanthine, sodium bicarbonate and 0.01% antifoam (Ucarferm™ Adjuvant 27,Union Carbide). Sterile SP-Sepharose beads were added to the fermenter.Upon termination of cell culture production in the fermenter, thesuspension, consisting of cells, cell debris and SP-Sepharose beads, isdispensed into sedimentation containers. The SP-Sepharose beads werewashed extensively in a series of buffers (20 mM sodium acetate/0.4Msodium chloride pH 4.0 and 20 mM sodium acetate/0.7M sodium chloride pH4.0) and 20 mM sodium acetate/1.0M sodium chloride pH 4.0 buffer. Analternative preferred buffer to prevent cell lysis is 0.15M NaCl at pH7.0 to remove intact cells followed by a higher NaCl concentrationbuffer to remove contaminants. The resulting monomer-enriched eluate ofthis step can be subjected to further processing as in Example 5, infra.

Elution of products remaining on the column in a single step with 1.5MNaCl-Acetate buffer provided a BPI dimer-rich (about 87%) eluate alsocontaining BPI multimers and monomers and potentially other contaminantssuch as nucleic acids, proteins including histones, and the like. Theeluate (2.6 liters) was diluted two-fold with water and was then loadedat 4.5 mL/minute onto a fresh 2.2×18.5 cm SP-Sepharose column which hadbeen equilibrated with 20 mM sodium acetate, 1.0M NaCl, pH, 4.0. Afterloading and washing, the column was reversed and eluted with 20 mMsodium acetate, 1.5M NaCl pH 4.0. The elution peak was collected in avolume of 70 mL and had a protein concentration of 0.67 mg/mL.

The SP-Sepharose eluate was diluted 3-fold to a salt concentration of0.5M with Milli-Q water and was then loaded at 2 mL/min onto a 1×15 cmCM-Sepharose column which had been equilibrated with 20 mM sodiumacetate, 0.5M NaCl, pH 4.0. After loading and washing at 0.5M NaCl toremove monomeric BPI products, the column was reversed and eluted with20 mM sodium acetate, 1.0M NaCl, pH 4.0 to provide a BPI protein productdimer-containing eluate. The elution peak was collected in a volume of15 mL and had a rBPI₂₃ concentration of 2.05 mg/mL.

The CM-Sepharose eluate was then further purified and buffer exchangedby passing it over a 2.2×20 cm Sephacryl S-100 column that had beenequilibrated with 20 mM sodium citrate, 0.15M NaCl, pH 5.0. The flowrate was 1.5 ml/minute and the product pool was collected in a volume of35 mL.

The concentration of dimer at this stage was 1.15 mg/mL. Excipients wereadded and the sample was diluted to give a final product at 1 mg/mL in20 mM sodium titrate, 0.15M NaCl, pH 5.0 with 0.1% poloxamer. 188 and0.002% polysorbate 80. This material was sterile filtered using a MillexGV 0.22 m filter into sterile 10 mL Hollister-Stier vials. Analysis ofthe final product indicated that it contained 40 mg dimer (50% yield)that was >99% pure. This is compared with the 1.5M NaCl-Acetatodimer-rich eluate from the first SP-Sepharose column which had a desiredprotein concentration of 18 μg/mL, 87% of which was dimer. DNA wasremoved throughout the various steps of the process but a significantproportion of the DNA was removed during elution from the SP-Sepharoseand CM-Sepharose columns where DNA was dissociated from the BPI proteinproducts at pH 4.0. Analysis of the dimeric product revealed that itpredominantly included monomers having BPI residues 1-199.

EXAMPLE 4 Comparison of Metal Ions as Dimer Formation Catalysts

Various metal ions (Cu²⁺, Ca²⁺, Zn²⁺, Mg²⁺ and Mn²⁺) were evaluated todetermine if they would promote the formation of BPI dimer.Specifically, 2 μL of ten-fold dilutions (1 mM, 0.1 mM and 0.01 mM) ofCuSO₄, ZnCl₂, CaCl₂, MgCl₂, and MnCl₂, were added into 18 μL of rBPI₂₃adjusted to pH 7.0 with 0.5M Tris, pH 10.6 and incubated for one hour at37° C. A 1 μg aliquot of each sample was then run on a 12.5% non-reducedSDS gel to determine the presence of dimer. The results showed dimerformation for the 1 mM and 0.1 mM Cu²⁺ samples but did not show dimerformation for the 0.01 mM Cu²⁺ or for the samples containing the otherions.

The experiment was repeated for ten-fold lower concentration (100 μM, 10μM and 1 μM) samples for each of the ions and the samples were incubatedfor 24 hours instead of 1 hour. The results indicated dimer formationonly for the 100 μM CuSO₄ sample which was the lowest concentrationgiving a positive result in the earlier example. This result suggeststhat the majority of dimer formation occurred in the first hour.

EXAMPLE 5 Conversion To Form Dimer

According to this example, a BPI protein product is converted to dimerby reaction in the presence of cupric sulfate (CuSO₄). A purified,formulated rBPI₂₃ product solution comprising 1 mg/mL rBPI₂₃ generallyisolated by practice of Steps A through C of Example 6, infra (andcontaining from 2 to 8 percent by weight dimeric product) in thepresence of 20 mM citrate pH 5.0, 150 mM NaCl, 0.1% poloxamer 188, and0.002% polysorbate 80 was exchanged into 50 mM Tris-HCl, 150 mM NaCl ,pH 8.0 by gel filtration using a Sephadex G-25 column. The BPI solutionwas then concentrated to about 1 mg/mL and sterile filtered withMillipore Millex GV 0.22 μM filters. CuSO₄ was added to variousconcentrations using either a 0.1 mM or a 1 mM stock solution. Sampleswere taken at defined intervals and the dimer conversion reaction wasquenched after 22 hours by running the samples over a Sephadex G-25column equilibrated in 20 mM citrate at pH 3.5 to remove the coppersulfate and reduce the reaction pH. The samples were then analyzed byion exchange (MA7C) HPLC to determine the relative proportions ofmonomer and dimer.

The results (FIG. 1) illustrate that the percent dimer formed frommonomer increases as a function of cupric sulfate concentration at pH8.0 but that at CuSO₄ concentrations above 5 μM little additional dimerwas formed at 22 hours. Results from other experiments indicate that therate of interchain disulfide formation is also a function of BPIconcentration. FIG. 2 depicts the results of a 24-hour time course offorming BPI dimer from the rBPI₂₃ monomer (1.0 mg/mL), at roomtemperature and either pH 7.0 or 8.0. The data is depicted as follows:--□--, % Dimer pH 7.0; . . .⋄. . . , % Dimer pH 8.0; . . .◯. . . , %Recovery pH 7.0; and . . .Δ. . . , % Recovery pH 8.0. The results showthat dimer formation proceeds more slowly at pH 7.0 (42% conversionafter 24 hours) than at pH 8.0 (85% conversion). The slower formation atpH 7.0 may be due to a lower concentration of cupric sulfate as a resultof copper precipitation in the presence of phosphate buffer.Nevertheless, the recovery of BPI dimer is higher at pH 7.0 than at pH8.0. This agrees with observations that BPI monomers and dimers are lessstable or soluble at higher pH values. Results from other experimentsindicated that the rate of dimer formation is greater at roomtemperature than at 4° C. while product recovery is greater at 4° C.than at room temperature. A comparison of the rates of dimer formationand protein recovery using Tris as opposed to phosphate buffersindicated little difference between the two.

The presence of CuSO₄ at 10 μM can accelerate the formation of dimerfrom monomer at pH 8 to greater than 85% in 5 hours if the startingmaterial is first reduced with DTT to eliminate cysteine adducts.Without reduction, greater than 75% of the monomer can be converted in 5hours.

EXAMPLE 6 Purification And Conversion to Form Dimer

A. Initial Isolation of Monomer

In this example, an alternative method for isolation of dimericmaterials from heterogeneous recombinant BPI protein productpreparations is presented. Specifically, transfected CHO cellsexpressing rBPI (1-193) were grown in the presence of SP-Sepharosebeads. Growth medium and SP-Sepharose resin were removed from rollerbottles, pooled and left for at least 15 minutes to allow theSP-Sepharose to settle to the bottom of the container. The bulk of themedium, clear of resin, was removed by decanting and then falteredthrough a device, such as a fritted disc, to permit the removal of cellsand the retention of the SP-Sepharose. Following the decanting of themedium, the SP-Sepharose was washed with 20 mM sodium acetate, pH 4.0,0.4M sodium chloride and washed again with 20 mM sodium acetate, pH 4.0,0.65M sodium chloride. An alternative preferred buffer to prevent celllysis is 0.15M NaCl at pH 7.0 to remove intact cells followed by ahigher NaCl concentration buffer to remove contaminants. TheSP-Sepharose was then eluted in a buffer comprising 20 mM sodiumacetate/acetic acid at pH 4.0 containing 1.0M NaCl, and 5 mM glycine ata flow rate of 100 cm/hour. The eluate was primarily monomeric BPI andthe BPI which remained on the column was primarily dimeric BPI.

B. Optional Virus Inactivation

The eluate was then subjected to an optional virus inactivation stepcomprising adjusting the eluate to pH 3.0 +/-0.1 with 10% hydrochloricacid and allowing it to stand at 2°-8° C. for approximately two hours.The pH was then adjusted to 4.0+/-0.1 with 1N sodium hydroxide anddiluted with water to 0.25M sodium chloride.

C. Concentration

The primarily monomeric BPI protein product preparation was thensubjected to a purification and concentration step wherein it was loadedonto a 1.6×10.0 cm CM-Sepharose column having a capacity of about 20 mLCM-Sepharose which had been pre-equilibrated with 20 mM sodium acetate,pH 4.0, 0.25 sodium chloride. The CM-Sepharose had a binding capacity of15 mg/mL BPI and the column was capable of a flow rate of 750 cm/hour.The column was washed with 30 column volumes of 20 mM sodium acetate, pH4.0, 0.35M sodium chloride and the BPI monomer was eluted with 20 mMsodium acetate, pH 8.7, 20 mM Tris, 0.6M sodium chloride.

D. Dimerization and Recovery

The BPI preparation was Falter sterilized and adjusted to pH 8.0 with 1Msodium hydroxide and a 1 mM stock solution of copper sulfate was addedto bring the CuSO₄ concentration to 10 μM. The preparation was thenincubated for 16-20 hours at room temperature to effect conversion todimer Alternatively, CuSO₄ could be added to a concentration of 30 μM ina 20 mM sodium acetate, 20 mM Tris, 0.6M NaCl solution at pH 8.0 and thepreparation incubated at 4° C. overnight. As a further alternative, thepreparation could be incubated with 30 μM CuSO₄ and 1M NaCl, 20 mMacetate at pH 5.0. After dimerization, the dimer/monomer ratio wasapproximately 3:1 by weight.

The pH of the resulting dimer-containing preparation was adjusted to 5.0with 10% HCl and two volumes of water and the solution was applied to a1×3 cm CM-Spherodex column having a volume of about 2.5 mL which hadbeen equilibrated with 20 mM sodium acetate, pH 5.0, 0.2 sodiumchloride. The CM-Spherodex had a binding capacity of 30 mg/mL BPI andthe column was capable of a flow rate of 300 cm/hour. The column waswashed with 10 column volumes of 20 mM sodium acetate, 0.5M sodiumchloride pH 5.0 to remove nonconverted monomer, and BPI dimer was theneluted with 20 mM sodium citrate, 1.0M sodium chloride, pH 5.0. TheCM-Sepharose and CM-Spherodex filtrations are believed to removesignificant levels of DNA and other impurities in the preparation. Forexample, DNA was removed during elution from the CM-Sepharose columnswhen DNA was dissociated from the BPI protein products at pH 4.0.

The CM-Spherodex dimer-containing eluate was applied to a 4.4×100 cmSephacryl S-100 column equilibrated with equilibration buffer (5 mMsodium citrate, 0.15M sodium chloride pH 5.0) and the column wasdeveloped with the equilibration buffer at about 6 mL/minute (21.6cm/hour). The resulting BPI product was greater than 95% dimer byweight.

EXAMPLE 7 Effects in a Rat Endotoxemia Model

In this example, a rat LPS infusion experimental endotoxemia model wasused to evaluate the effects of formulations of rBPI₂₃, rBPI₂₁ Δcys, andBPI dimer on serum TNF levels. Groups of 4-10 rats were anesthetized andwere subjected to intravenous infusion into the jugular vein with E.coli 0111:B4 endotoxin at a dosage of 0.25 mg/kg over a 30 minuteperiod. Each rat was simultaneously infused into the femoral vein withvarious concentrations of test compound or the buffer vehicle as acontrol. The animals were then bled from the femoral vein at varioustimes for a TNF determination using a standard assay with the resultsshown in FIG. 3. The data is depicted as follows: --□--, vehicle;---▴---, rBPI₂₁ Δcys (3 mg/kg); --Δ--, BPI dimer (0.1 mg/kg); ----,rBPI₂₃ (0.3 mg/kg); ---Δ---, rBPI₂₁ Δcys (10 mg/kg); --◯--, rBPI₂₃ (3mg/kg); ---♦---, rBPI₂₁ Δcys (20 mg/kg); --▪-- BPI dimer (1 mg/kg).Specifically, BPI dimer (0.1 mg/kg), rBPI₂₃ (0.3 mg/kg), rBPI₂₁ Δcys (10mg/kg), rBPI₂₃ (3 mg/kg) and rBPI₂₁ Δcys (20 mg/kg) were allsignificantly different from vehicle and rBPI₂₁ Δcys (3 mg/kg) but notsignificantly different from each other. Effects of BPI dimer (1 mg/kg)were significantly differera from all groups except for rBPI₂₁ Δcys (20mgacg). As observed with other endotoxemia models TNFα levels peaked 90minutes after endotoxin infusion began and then returned toward controllevels. Dosages of 0.3 to 3.0 mg/kg of rBPI₂₃ resulted in approximatelya 60% reduction in TNF levels compared to the buffer treated animals.Dosages of 10-20 mg/kg rBPI₂₁ Δcys were required to reduce TNF levels tothe same degree as rBPI₂₃ (3.0 mg/kg). In contrast, a dosage of BPIdimer at 0.1 mg/kg significantly inhibited TNF release. This level ofinhibition was comparable to that achieved with 0.3 mg/kg of rBPI₂₃ or10 mg/kg of rBPI₂₁ Δcys.

The observed enhanced in vivo biological activities of formulationsincluding dimeric BPI protein product forms were particularly unexpectedin view of in vitro assays wherein activities of products with highconcentrations of dimeric molecular forms were not significantlydifferent from those with lesser concentrations. Specifically, in an exvivo cytokine assay for TNF production where E. coli O113 LPS wasincubated in the presence of human peripheral blood BPI dimer inhibitedTNF production with similar potency to that of monomer when compared ona molar basis. In a broth dilution antibacterial assay BPI dimer andmonomer were equally potent on a molar basis against E. coli J5. In aradial diffusion assay against E. coli J5 BPI dimer appeared to be lessactive than monomer. The lower antibacterial activity of dimer in theradial diffusion assay may be an artifact due to the lower diffusion ofdimer through the agarose. because a Coomassie Blue-stained halo wasobserved around the wells for the dimer but not the monomer.

EXAMPLE 8 Effect of Dimer in a Rabbit Endotoxemia Model

In this example, a conscious rabbit endotoxemia model was used toevaluate the effects of various BPI protein products on cardiopulmonaryfunction. These BPI protein products included rBPI23, rBPI21Δcys anddimeric.

Rabbits under anesthesia were instrumented for measurement of bloodpressure, heart rate, and cardiac output. After recovery from anesthesia6 μg/kg of E. coli 0113 endotoxin was injected into a marginal ear veinover a 60 second period. Test products at dosages of from 0.54-4 mg/kgwere injected into a jugular vein over a 120 second period beginning 30seconds before administration of endotoxin. Cardiovascular andrespiratory parameters were then measured for a 3 hour period and bloodsamples were obtained periodically in order to measure blood gases.

The results illustrated in FIG. 4 disclose the effect of the BPIproteins compared with a buffer control on mean arterial blood pressurein the rabbit endotoxemia model. Blood pressure of the animals decreasedafter endotoxin challenge and reached levels close to 50 mmHg by 60minutes into the experiment. It required 4 mg/kg rBPI₂₁ Δcys to preventthe decrease in blood pressure and to maintain pressure in the normalrange of about 80-100 mmHg throughout the experiment. A dosage of 2mg/kg of rBPI₂₃ was sufficient to prevent decreases in blood pressureoutside of the normal range throughout the experiment. In contrast, adosage of only 0.5 mg/kg of the dimer was required to prevent decreasesin blood pressure outside of the normal range throughout the experiment.

Evaluation of changes in cardiac index indicated that the cardiac indexwas significantly reduced in animals treated only with the buffercontrol. The decrease in cardiac index was reduced or eliminated withtreatment of BPI proteins. Specifically a dosage of 4 mg/kg of rBPI₂₁Δcys, 2 mg/kg rBPI₂₃, and 0.5 mg/kg BPI dimer neutralized the effect ofendotoxemia on cardiac output. Similar neutralization effects wereobserved with respect to decreases in total peripheral resistance, withrespect to elevated heart rate, and with respect to elevated respiratoryrate (see FIG. 5).

EXAMPLE 9 Compantive Activity of Reduced (Monomeric) rBPI₂₃ in a RatEndotoxemia Model

In this example, the activity of an rBPI₂₃ preparation substantiallyfree of dimer was determined in a rat experimental endotoxemia model. Inthis manner, the overall contribution to biological activity (such asobserved in Example 8) of BPI dimer component present in fermenterproduced rBPI₂₃ could be determined. Specifically, the monomericpreparation was produced by treatment of fermenter produced rBPI₂₃ with20 mM dithiothreitol (DTF) at pH 7.0 for 30 minutes at room temperature.A study using the rat LPS infusion model according to Example 7 was thenconducted to determine the activity of the product. Groups of five ratseach were simultaneously infused (30 minutes, i.v.) with 0111:B4 LPS(0.25 mg/kg) and either DTT-reduced rBPI₂₃ (0.3, 3.0 or 10.0 mg/kg),rBPI₂₁ Δcys (3.0 or 10.0 mg/kg) or vehicle. The reducing agent waspresent in all preparations.

Serum was collected at 90 minutes and assayed for TNF with the meanvalue results shown in FIG. 6. None of the BPI-treated groups had TNFlevels that were statistically significantly lower than the vehiclecontrol group. This was due to the considerable variability in thecontrol group in this study and the small number of animals per group.Nevertheless, there was a trend toward inhibition of TNF release at a 10mg/kg dose of rBPI₂₁ Δcys. The magnitude of the inhibition was similarto that obtained with this dose in Example 7. There was also a trendtoward inhibition with the 10 mg/kg dose of the monomeric preparation.Therefore, the monomeric preparation appeared to have a similar potencyto the rBPI₂₁ Δcys. Moreover, the monomeric preparation apparently has alower potency than non-reduced rBPI₂₃ which has been shown to beeffective at a dose as low as 0.3 mg/kg (e.g., Example 7). These resultssuggest that the presence of BPI dimer in preparations of rBPI₂₃ mayaccount for its increased potency relative to rBPI₂₁ Δcys.

EXAMPLE 10 Effect of Dimer in a Mouse Peritonitis Model

In this example, the efficacy of BPI dimer and fermenter produced rBPI₂₃was determined in a mouse model of acute peritonitis. Groups of 15 micewere challenged with 10⁷ live E. coli bacteria (strain 07:K1) in 0.5 mLvolumes and then treated with 20, 50, or 100 μg of dimer, or rBPI₂₃ in1mL volumes or an equal volume of buffer. The animals were observed for7 days and mortality recorded. FIG. 7 shows the final number ofsurvivors at each dose with the 0 dose representing the mice givenbuffer. While the effects of the high doses of the constructs were notsignificantly different, the 20 μg dose of BPI dimer was significantlymore protective than the same dose of rBPI₂₃.

EXAMPLE 11 Effects in a Mouse Endotoxemia Model

In this example, BPI dimer was tested for its efficacy in a mouseexperimental endotoxemia model. Groups of 15 mice were administered anintravenous injection of endotoxin (E. coli O111:B4, Sigma Chemical Co.,St. Louis, Mo.) at a LD₉₀ dosage of 40 mg/kg. This was followed by asecond intravenous injection of the BPI dimer or rBPI₂₁ Δcys. Injectionsof buffer were used in negative control mice. The animals were observedfor 7 days and mortality recorded. A 10 mg/kg dose of BPI dimer waseffective to protect all treated animals (100% survival). A 30 mg/kgdose of rBPI₂₁ Δcys was required to similarly protect the treated mice.No animals survived in the buffer control group.

EXAMPLE 12 Pharmacokinetics of BPI Dimer in Rats

In this example, the pharmacokinetics of BPI dimer in rats was comparedwith that of fermenter produced rBPI₂₃. Rats were given intravenousbolus injections with 1 mg/kg of either fermenter produced rBPI₂₃ or BPIdimer. BPI levels were then determined in plasma samples taken from therats at various time points by ELISA assay. The results shown in FIG. 14depict that rBPI₂₃ and BPI dimer both cleared rapidly from the plasmawith systemic mean residence times of 1-3 minutes, but theirdistribution patterns differed considerably: the steady state volume ofdistribution for BPI dimer was in the range of about 700-1100 mL/kgversus 70-120 mL/kg for rBPI₂₃.

Western blot analysis conducted of serum of rats into which BPI dimerhad been injected found no detectable monomer indicating that there isnegligible break down of BPI dimer in vivo.

EXAMPLE 13 Binding of LPS to BPI Protein Products Pre-Bound to HumanEndothelial Cells

In this example, rBPI₂₃, rBPI₂₁ Δcys, and BPI dimer were compared withregard to their abilities to bind LPS after the BPI protein productswere prebound to human endothelial cells. A "sandwich experiment" wasperformed in which confluent human umbilical vein endothelial cells(HUVEC) were first incubated with various BPI protein products, thecultures washed, and then incubated with labelled LPS molecules.

Specifically, HUVEC cells were obtained from Clonetics (San Diego,Calif.) and cultured for 5 to 12 passages in Endothelial Cell GrowthMedium (EGM-UV) purchased from Clonetics. The HUVEC cells were grown in24-well microtiter plates (seeding density=5×10⁴ cells/well) in EGM-UV,for 3 days in order to get confluent monolayers. The HUVEC cells werewashed twice in PBS/0.1% BSA (PBS/BSA) and incubated in the presence of0.75 μg or 1.5 μg of the test products for 3 hours at 4° C. in 500 μlPBS/BSA. Following incubation, the cells were washed twice with coldPBS/BSA and incubated with 500 μL PBS/BSA containing ¹²⁵ I-RaLPS(231,000 cpm=50 ng/well) with or without 1000-fold excess unlabeledRaLPS (50 μg/well) for an additional 2.5 hours at 4° C. The cells werethen washed 3 times with PBS/BSA, solubilized with 500 μl of 1M sodiumhydroxide and the lysates counted in a gamma counter. The binding of ¹²⁵I-RaLPS in the presence of 1000 fold excess unlabeled RaLPS was taken torepresent non-specific binding with specific binding of ¹²⁵ I-RaLPSdefined as the difference between total and non-specific binding. Theresults are shown in FIG. 8 with the amount of RaLPS bound expressed asng/well. Those results indicate that only dimer retained the ability tobind ¹²⁵ I-LPS when it was bound to HUVEC cells while rBPI₂₃ and rBPI₂₁Δcys did not.

The procedure described above was essentially reproduced with theexception that the HUVEC cells were incubated in the presence of 1 μg or3 μg of the various BPI protein products (including rBPI₂₃, recombinantBPI holoprotein and BPI dimer) for 3 hours at 4° C. in 500 μl PBS/BSAcontaining ¹²⁵ I-RaLPS (340,000 cpm=73 ng/well) for an additional 2.5hours at 4° C. The cells were then washed 3 times with PBS/BSA,solubilized with 500 μl of 1M sodium hydroxide and the lysates countedin a gamma counter. The results in FIG. 9 show high levels of binding bythe BPI dimer. Consistent with the results of the earlier procedure,there was no significant binding of ¹²⁵ I-LPS by rBPI₂₃ at aconcentration of 1 μg/well, but there was some binding at aconcentration of 3 μwell. This may be a consequence of the presence oflow levels of dimer in the rBPI₂₃ preparation. The rBPI holoproteinfailed to bind ¹²⁵ I-LPS to any significant extent. Furtherexperimentation indicated that BPI dimer is bound to HUVEC cells toabout the same degree as fermenter produced rBPI₂₃ and rBPI₂₁ Δcys. Thissuggests that the dramatically enhanced ability of BPI dimer to bind toLPS in these assays is not due to the greater binding of dimer to thecells. Still further experimentation established that dimer which isbound to HUVEC cells at physiological temperatures (37° C. rather than4° C.) retains its ability to bind ¹²⁴ I-RaLPS.

EXAMPLE 14 Heparin Binding by BPI Protein Products

The capacity of BPI protein products including BPI dimer to bind toheparin was determined using membrane bound natural and recombinant BPImolecules and radiolabelled heparin. Briefly, rBPI₂₃ and BPI dimer wereadded to wells of a 96-well microtiter plate having an Immobilon-P(Millipore, Bedford, Mass.) membrane disposed at the bottom of thewells. One μg of protein was added to each well. The wells were driedand subsequently blocked with a 0.1% bovine serum albumin (BSA) inphosphate buffered saline, pH 7.4 (blocking buffer.) Dilutions of ³H-heparin (DuPont, NEN, Wilmington, Del.) were made in the blockingbuffer and incubated in the BPI containing wells for one hour at 4° C.The unbound heparin is aspirated and the wells were washed three timeswith blocking buffer, dried and removed for quantitation in a liquidscintillation counter. Typical assay results are graphically presentedin FIG. 10. These results show that BPI dimer has significantly greaterheparin binding than does the monomer. While BSA in the blocking bufferdoes have a low affinity and capacity to bind heparin, this wasconsidered physiologically irrelevant and the background was routinelysubtracted from the test compound signal.

In addition, binding constants with ³ H-heparin as the ligand weredetermined using nonlinear function minimization with Grafit software(Erithicus Softward Ltd., Staines, UK) for rBPI₂₃, and BPI dimer withthe results shown in Table 2 below.

                  TABLE 2                                                         ______________________________________                                        Binding Constants with .sup.3 H-heparin as the Ligand                         PROTEIN     K.sub.d     CAPACITY                                              ______________________________________                                        rBPI.sub.23 274 nM +/- 0.44                                                                             244 ng +/- 3.7                                      BPI Dimer   345 nM +/- 0.42                                                                           293.3 ng +/- 3.3                                      ______________________________________                                    

EXAMPLE 15 Heparin Neutralization by BPI Protein Products: Effect of BPIDimer on Heparin-Mediated Lengthening of Thrombin Time In Vitro

The effect of BPI protein products on heparin-mediated lengthening ofthrombin time, i.e., the time required for clotting of a mixture ofthrombin and plasma was examined. Thrombin time is lengthened by thepresence of endogenous or exogenous inhibitors of thrombin formation,such as therapeutically administered heparin. Agents which neutralizethe anti-coagulant effects of heparin will reduce the thrombin timemeasured by the test. Human citrated plasma (200 μL) was incubated for 1minute at 37° C. with either 15 μL of diluent (0.15M NaCl, 0.1M Tris, pH7.4) or 15 μL of the diluent also containing 25 μg/mL heparin (187units/mg). Various concentrations of BPI dimer and rBPI₂₁ Δcys involumes of 15 μL were added, followed immediately by 100 μL of thrombinreagent (Sigma Chemical Co., No. 845-4). Clotting time (thrombin time)was measured using a BBL Fibrometer (Becton Diekenson MicrobiologySystems, Cockeysville, Md.). The results shown in FIG. 11 establish thatboth BPI protein products inhibit the heparin-mediated lengthening ofthrombin time but that the BPI dimer does so at significantly lowerweight concentrations.

EXAMPLE 16 Heparin Neutralization by BPI Protein Products: Effect of BPIDimer on Heparin-Mediated Lengthening of Activated PartialThromboplastin Time In Vivo

The effect of BPI protein products on heparin-mediated lengthening ofactivated partial thromboplastin time (aPTT), i.e., the time requiredfor clotting of a mixture of thrombin and plasma was examined. aPTT islengthened by the presence of exogenous inhibitors of thrombinformation, such as therapeutically administered heparin. Agents whichneutralize the anti-coagulant effects of heparin will reduce the aPTTtime measured by the test. Human titrated plasma (200 μL) was incubatedfor 1 minute at 37° C. with either 15 μL of diluent (0.15M NaCl, 0.1MTris, pH 7.4) or 15 μL of the diluent also containing 25 μg/mL heparin(187 units/mg). Various concentrations of fermenter produced rBPI₂₃, BPIdimer, and rBPI₂₁ Δcys in a volume of 15 μL were added, followedimmediately by 100 μL of thrombin reagent (Sigma Chemical Co., No.845-4). aPTT was measured using a BBL Fibrometer (Becton DickensonMicrobiology Systems, Cockeysville, Md.) with the results shown in FIG.12. The data is depicted as follows: ------------, rBPI₂₃ ;------◯------, rBPI₂₁ Δcys; ------▪------, BPI dimer; ------Δ------, PBSbuffer; ------▴------, no heparin. Specifically, the data establish thatboth rBPI₂₃ and rBPI₂₁ Δcys inhibit the heparin-mediated lengthening ofaPTT but that BPI dimer does so at substantially lower concentrations.

The effect of BPI dimer on partial thromboplastin time (aPTT) inheparinized rats was determined. aPTT is lengthened by the presence ofendogenous or exogenous inhibitors of thrombin formation, such astherapeutically administered heparin. Agents which neutralize theanti-coagulant effects of heparin will reduce the aPTT as measured bythe test. Sprague-Dawley rats housed under NIH guidelines andanesthetized by Metaphane® inhalation were administered with 100 U/kgheparin by bolus intravenous injections via the tail vein followed tenminutes later by intravenous administration of 0.5 or 1.0 mg/kg BPIdimer, or 1 mg/kg thaumatin control protein (having charge and sizesimilar to rBPI₂₃). Five minutes later, blood samples were collectedfrom the abdominal aorta, plasma was separated therefrom and the aPTTwas then determined. The aPTF of a group of non-heparinized PBS treatedanimals treated with 1 mg/kg rBPI₂₃ was also determined. The resultsshown in FIG. 13 establish that the BPI dimer significantly reduced theaPTT of the treated animals. These animal data confirm, in vivo, theheparin neutralizing effects of BPI dimer as shown above.

EXAMPLE 17 Heparin Neutralization by BPI Protein Products: Effect of BPIin a Matrige™ Model of Angiogenesis

In this example, the ability of BPI dimer to neutralize heparin in aMatrigel™ model of angiogenesis is determined. The Matrigel™ modeldescribed by Passaniti et al., Lab. Invest. 67:519-528 (1992) uses abasement membrane preparation mixed with FGF 1 or FGF 2 and heparin (40U/mL). The mixture induces an intense vascular response when injectedsubcutaneously into mice and the extent of angiogenesis is quantitatedby measurement of the hemoglobin content of the gels. Compounds whichneutralize the angiogenic properties of heparin will inhibitangiogenesis in the Matrigel™ model.

Specifically, Matrigel™ (Collaborative Biomedical Products) ismaintained at 4° C. as angiogenic factors are added to the gel in itsliquid state. Heparin Sodium (is dissolved in sterile phosphate-bufferedsaline to various concentrations from 10,000-1,250 U/mL. RecombinantbhFGF (BACHEM Bioscience, Inc.) is diluted with sterile PBS to 200ng/mL. A volume of 2.5 μL dissolved Heparin Sodium and 2.5 μLrecombinant bhFGF is added to 0.5 mL Matrigel™ per mouse injection. BPIdimer is added at 10 μL (1 mg/mL) per 0.5 ml Matrigel™. Sterile PBS isadded at 10 μL per injection to those Matrigel™ mixtures not comprisingthe BPI dimeL Prepared Matrigel™ mixtures are vortexed and drawn intopre-cooled syringes.

Male C57BL/6J mice (Jackson Lab, Bar Harbor, Me.) are 6-8 weeks of agewhen Matrigel™ mixtures are subcutaneously injected with 0.5 mL of themixture near the abdominal midline. Seven days subsequent to injectionsgels are excised and placed in 500 μL Drabkin's reagent. Total proteinand hemoglobin content are determined for the gels stored in Drabkin'sreagent after homogenation of the gels.

Numerous modifications and variations in the practice of the inventionare expected to occur to those skilled in the an upon consideration ofthe presently preferred embodiments thereof. Consequently, the onlylimitations which should be placed upon the scope of the invention arethose which appear in the appended claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 11                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1813 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 31..1491                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: mat_peptide                                                     (B) LOCATION: 124..1491                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       CAGGCCTTGAGGTTTTGGCAGCTCTGGAGGATGAGAGAGAACATGGCCAGGGGC54                      MetArgGluAsnMetAlaArgGly                                                      31-30-25                                                                      CCTTGCAACGCGCCGAGATGGGTGTCCCTGATGGTGCTCGTCGCCATA102                           ProCysAsnAlaProArgTrpValSerLeuMetValLeuValAlaIle                              20-15- 10                                                                     GGCACCGCCGTGACAGCGGCCGTCAACCCTGGCGTCGTGGTCAGGATC150                           GlyThrAlaValThrAlaAlaValAsnProGlyValValValArgIle                              515                                                                           TCCCAGAAGGGCCTGGACTACGCCAGCCAGCAGGGGACGGCCGCTCTG198                           SerGlnLysGlyLeuAspTyrAlaSerGlnGlnGlyThrAlaAlaLeu                              10152025                                                                      CAGAAGGAGCTGAAGAGGATCAAGATTCCTGACTACTCAGACAGCTTT246                           GlnLysGluLeuLysArgIleLysIleProAspTyrSerAspSerPhe                              303540                                                                        AAGATCAAGCATCTTGGGAAGGGGCATTATAGCTTCTACAGCATGGAC294                           LysIleLysHisLeuGlyLysGlyHisTyrSerPheTyrSerMetAsp                              455055                                                                        ATCCGTGAATTCCAGCTTCCCAGTTCCCAGATAAGCATGGTGCCCAAT342                           IleArgGluPheGlnLeuProSerSerGlnIleSerMetValProAsn                              606570                                                                        GTGGGCCTTAAGTTCTCCATCAGCAACGCCAATATCAAGATCAGCGGG390                           ValGlyLeuLysPheSerIleSerAsnAlaAsnIleLysIleSerGly                              758085                                                                        AAATGGAAGGCACAAAAGAGATTCTTAAAAATGAGCGGCAATTTTGAC438                           LysTrpLysAlaGlnLysArgPheLeuLysMetSerGlyAsnPheAsp                              9095100105                                                                    CTGAGCATAGAAGGCATGTCCATTTCGGCTGATCTGAAGCTGGGCAGT486                           LeuSerIleGluGlyMetSerIleSerAlaAspLeuLysLeuGlySer                              110115120                                                                     AACCCCACGTCAGGCAAGCCCACCATCACCTGCTCCAGCTGCAGCAGC534                           AsnProThrSerGlyLysProThrIleThrCysSerSerCysSerSer                              125130135                                                                     CACATCAACAGTGTCCACGTGCACATCTCAAAGAGCAAAGTCGGGTGG582                           HisIleAsnSerValHisValHisIleSerLysSerLysValGlyTrp                              140145150                                                                     CTGATCCAACTCTTCCACAAAAAAATTGAGTCTGCGCTTCGAAACAAG630                           LeuIleGlnLeuPheHisLysLysIleGluSerAlaLeuArgAsnLys                              155160165                                                                     ATGAACAGCCAGGTCTGCGAGAAAGTGACCAATTCTGTATCCTCCAAG678                           MetAsnSerGlnValCysGluLysValThrAsnSerValSerSerLys                              170175180185                                                                  CTGCAACCTTATTTCCAGACTCTGCCAGTAATGACCAAAATAGATTCT726                           LeuGlnProTyrPheGlnThrLeuProValMetThrLysIleAspSer                              190195200                                                                     GTGGCTGGAATCAACTATGGTCTGGTGGCACCTCCAGCAACCACGGCT774                           ValAlaGlyIleAsnTyrGlyLeuValAlaProProAlaThrThrAla                              205210215                                                                     GAGACCCTGGATGTACAGATGAAGGGGGAGTTTTACAGTGAGAACCAC822                           GluThrLeuAspValGlnMetLysGlyGluPheTyrSerGluAsnHis                              220225230                                                                     CACAATCCACCTCCCTTTGCTCCACCAGTGATGGAGTTTCCCGCTGCC870                           HisAsnProProProPheAlaProProValMetGluPheProAlaAla                              235240245                                                                     CATGACCGCATGGTATACCTGGGCCTCTCAGACTACTTCTTCAACACA918                           HisAspArgMetValTyrLeuGlyLeuSerAspTyrPhePheAsnThr                              250255260265                                                                  GCCGGGCTTGTATACCAAGAGGCTGGGGTCTTGAAGATGACCCTTAGA966                           AlaGlyLeuValTyrGlnGluAlaGlyValLeuLysMetThrLeuArg                              270275280                                                                     GATGACATGATTCCAAAGGAGTCCAAATTTCGACTGACAACCAAGTTC1014                          AspAspMetIleProLysGluSerLysPheArgLeuThrThrLysPhe                              285290295                                                                     TTTGGAACCTTCCTACCTGAGGTGGCCAAGAAGTTTCCCAACATGAAG1062                          PheGlyThrPheLeuProGluValAlaLysLysPheProAsnMetLys                              300305310                                                                     ATACAGATCCATGTCTCAGCCTCCACCCCGCCACACCTGTCTGTGCAG1110                          IleGlnIleHisValSerAlaSerThrProProHisLeuSerValGln                              315320325                                                                     CCCACCGGCCTTACCTTCTACCCTGCCGTGGATGTCCAGGCCTTTGCC1158                          ProThrGlyLeuThrPheTyrProAlaValAspValGlnAlaPheAla                              330335340345                                                                  GTCCTCCCCAACTCCTCCCTGGCTTCCCTCTTCCTGATTGGCATGCAC1206                          ValLeuProAsnSerSerLeuAlaSerLeuPheLeuIleGlyMetHis                              350355360                                                                     ACAACTGGTTCCATGGAGGTCAGCGCCGAGTCCAACAGGCTTGTTGGA1254                          ThrThrGlySerMetGluValSerAlaGluSerAsnArgLeuValGly                              365370375                                                                     GAGCTCAAGCTGGATAGGCTGCTCCTGGAACTGAAGCACTCAAATATT1302                          GluLeuLysLeuAspArgLeuLeuLeuGluLeuLysHisSerAsnIle                              380385390                                                                     GGCCCCTTCCCGGTTGAATTGCTGCAGGATATCATGAACTACATTGTA1350                          GlyProPheProValGluLeuLeuGlnAspIleMetAsnTyrIleVal                              395400405                                                                     CCCATTCTTGTGCTGCCCAGGGTTAACGAGAAACTACAGAAAGGCTTC1398                          ProIleLeuValLeuProArgValAsnGluLysLeuGlnLysGlyPhe                              410415420425                                                                  CCTCTCCCGACGCCGGCCAGAGTCCAGCTCTACAACGTAGTGCTTCAG1446                          ProLeuProThrProAlaArgValGlnLeuTyrAsnValValLeuGln                              430435440                                                                     CCTCACCAGAACTTCCTGCTGTTCGGTGCAGACGTTGTCTATAAA1491                             ProHisGlnAsnPheLeuLeuPheGlyAlaAspValValTyrLys                                 445450455                                                                     TGAAGGCACCAGGGGTGCCGGGGGCTGTCAGCCGCACCTGTTCCTGATGGGCTGTGGGGC1551              ACCGGCTGCCTTTCCCCAGGGAATCCTCTCCAGATCTTAACCAAGAGCCCCTTGCAAACT1611              TCTTCGACTCAGATTCAGAAATGATCTAAACACGAGGAAACATTATTCATTGGAAAAGTG1671              CATGGTGTGTATTTTAGGGATTATGAGCTTCTTTCAAGGGCTAAGGCTGCAGAGATATTT1731              CCTCCAGGAATCGTGTTTCAATTGTAACCAAGAAATTTCCATTTGTGCTTCATGAAAAAA1791              AACTTCTGGTTTTTTTCATGTG1813                                                    (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 487 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetArgGluAsnMetAlaArgGlyProCysAsnAlaProArgTrpVal                              31-30-25-20                                                                   SerLeuMetValLeuValAlaIleGlyThrAlaValThrAlaAlaVal                              15-10-51                                                                      AsnProGlyValValValArgIleSerGlnLysGlyLeuAspTyrAla                              51015                                                                         SerGlnGlnGlyThrAlaAlaLeuGlnLysGluLeuLysArgIleLys                              202530                                                                        IleProAspTyrSerAspSerPheLysIleLysHisLeuGlyLysGly                              354045                                                                        HisTyrSerPheTyrSerMetAspIleArgGluPheGlnLeuProSer                              50556065                                                                      SerGlnIleSerMetValProAsnValGlyLeuLysPheSerIleSer                              707580                                                                        AsnAlaAsnIleLysIleSerGlyLysTrpLysAlaGlnLysArgPhe                              859095                                                                        LeuLysMetSerGlyAsnPheAspLeuSerIleGluGlyMetSerIle                              100105110                                                                     SerAlaAspLeuLysLeuGlySerAsnProThrSerGlyLysProThr                              115120125                                                                     IleThrCysSerSerCysSerSerHisIleAsnSerValHisValHis                              130135140145                                                                  IleSerLysSerLysValGlyTrpLeuIleGlnLeuPheHisLysLys                              150155160                                                                     IleGluSerAlaLeuArgAsnLysMetAsnSerGlnValCysGluLys                              165170175                                                                     ValThrAsnSerValSerSerLysLeuGlnProTyrPheGlnThrLeu                              180185190                                                                     ProValMetThrLysIleAspSerValAlaGlyIleAsnTyrGlyLeu                              195200205                                                                     ValAlaProProAlaThrThrAlaGluThrLeuAspValGlnMetLys                              210215220225                                                                  GlyGluPheTyrSerGluAsnHisHisAsnProProProPheAlaPro                              230235240                                                                     ProValMetGluPheProAlaAlaHisAspArgMetValTyrLeuGly                              245250255                                                                     LeuSerAspTyrPhePheAsnThrAlaGlyLeuValTyrGlnGluAla                              260265270                                                                     GlyValLeuLysMetThrLeuArgAspAspMetIleProLysGluSer                              275280285                                                                     LysPheArgLeuThrThrLysPhePheGlyThrPheLeuProGluVal                              290295300305                                                                  AlaLysLysPheProAsnMetLysIleGlnIleHisValSerAlaSer                              310315320                                                                     ThrProProHisLeuSerValGlnProThrGlyLeuThrPheTyrPro                              325330335                                                                     AlaValAspValGlnAlaPheAlaValLeuProAsnSerSerLeuAla                              340345350                                                                     SerLeuPheLeuIleGlyMetHisThrThrGlySerMetGluValSer                              355360365                                                                     AlaGluSerAsnArgLeuValGlyGluLeuLysLeuAspArgLeuLeu                              370375380385                                                                  LeuGluLeuLysHisSerAsnIleGlyProPheProValGluLeuLeu                              390395400                                                                     GlnAspIleMetAsnTyrIleValProIleLeuValLeuProArgVal                              405410415                                                                     AsnGluLysLeuGlnLysGlyPheProLeuProThrProAlaArgVal                              420425430                                                                     GlnLeuTyrAsnValValLeuGlnProHisGlnAsnPheLeuLeuPhe                              435440445                                                                     GlyAlaAspValValTyrLys                                                         450455                                                                        (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       AAGCTTGTCGACCAGGCCTTGAGGT25                                                   (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       CTGGAGGCGGTGATGGTG18                                                          (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       CTCCAGCAGCCACATCAAC19                                                         (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       GAACTTGGTTGTCAGTCG18                                                          (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       GCCACCRCCATGG13                                                               (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       ACTGTCGACGCCACCATGGCCAGGGGC27                                                 (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       CCGCGGCTCGAGCTATATTTTGGTCAT27                                                 (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      CTGTAGCTCGAGCCGC16                                                            (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      GGCTCGAGCTACAGAGT17                                                           __________________________________________________________________________

We claim:
 1. A method of therapeutic use of abactericidal/permeability-increasing protein product, wherein theimprovement comprises advertising to a patent abactericidal/permeability-increasing protein product formulationcontaining greater than 50 percent of said product in the form of astable covalently or non-covalently linked dimeric product characterizedby enhanced in vivo biological activity in comparison to the monomericform of said product.
 2. The improvement of claim 1 wherein said productformulation contains greater than 75 percent dimericbactericidal/permeability-increasing protein product.
 3. The improvementof claim 1 wherein said product formulation contains greater than 90percent dimeric bactericidal/permeability-increasing protein product. 4.The improvement of claim 1 wherein said product formulation containsgreater than 95 percent dimeric bactericidal/permeability-increasingprotein product.
 5. The improvement of claim 1 wherein the monomericbactericidal/permeability-increasing protein product is full lengthbactericidal/permeability-increasing protein.
 6. Abactericidal/permeability-increasing protein product pharmaceuticalcomposition containing greater than 50 percent of a stable covalently ornon-covalently linked dimeric product characterized by an enhanced invivo biological activity in comparison to the monomeric form of saidproduct and a pharmaceutically acceptable diluent, adjuvant or carrier.7. A bactericidal/permeability-increasing protein product pharmaceuticalcomposition containing greater than 75 percent of a stable covalently ornon-covalently linked dimeric product characterized by an enhanced invivo biological activity in comparison to the monomeric form of saidproduct and a pharmaceutically acceptable diluent, adjuvant or carrier.8. A bactericidal/permeability-increasing protein product pharmaceuticalcomposition containing greater than 90 percent of a stable covalently ornon-covalently linked dimeric product characterized by an enhanced invivo biological activity in comparison to the monomeric form of saidproduct and a pharmaceutically acceptable diluent, adjuvant or carrier.9. A bactericidal/permeability-increasing protein product pharmaceuticalcomposition containing greater than 95 percent of a stable covalently ornon-covalently linked dimeric product characterized by an enhanced invivo biological activity in comparison to the monomeric form of saidproduct and a pharmaceutically acceptable diluent, adjuvant or carrier.